Compositions and methods comprising anti-nrp2 antibodies

ABSTRACT

Provided are antibodies and antigen-binding fragments thereof that specifically bind to human neuropilin-2 (NRP2) polypeptides, including those that modulate binding interactions between human NRP2 and at least one NRP2 ligand, for example, human histidyl-tRNA synthetase (HRS), and which thereby modulate subsequent NRP2-mediated downstream signaling events, including related therapeutic compositions and methods for modulating NRP2 activity and treating diseases such as NRP2-associated diseases

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Patent Application No. 62/653,823, filed Apr. 6, 2018, which isincorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ATYR_134_01 US_ST25.txt. The text file is 228KB, created on Apr. 5, 2019, and is being submitted electronically viaEFS-Web.

BACKGROUND Technical Field

Embodiments of the present disclosure relate to antibodies andantigen-binding fragments thereof that specifically bind to humanneuropilin-2 (NRP2) polypeptides, including those that modulate bindinginteractions between human NRP2 and at least one NRP2 ligand, forexample, human histidyl-tRNA synthetase (HRS), and which therebymodulate subsequent NRP2-mediated downstream signaling events, includingrelated therapeutic compositions and methods for modulating NRP2activity and treating diseases such as NRP2-associated diseases.

Description of the Related Art

Recent research developments suggest that tRNA synthetases playimportant roles in cellular responses beyond their well-characterizedrole in protein synthesis. In particular, there is a growing recognitionthat tRNA synthetases participate in a range of previously-unrecognizedroles in responding to cellular stress and tissue homeostasis, in bothintracellular and extracellular environments.

The Resokine family of proteins (HRS Polypeptides) are derived from thehistidyl-tRNA synthetase gene (HARS) via proteolysis or alternativesplicing, and are important modulators of both intracellular andextracellular activity. Extracellular HARS is readily detectable in thecirculation in normal healthy volunteers, and autoantibodies to HARS(Jo-1 antibodies) have been characterized in inflammatory myopathies(IM) and in subjects with inflammatory lung disease (ILD). While therole of Jo-1 antibodies in disease progression is not yet wellunderstood, subjects with Jo-1 antibodies tend to be less susceptible tocancer compared to subjects with inflammatory myopathies without Jo-1antibodies (see, e.g., Lu et al., PLOS ONE 9(4) e94128, 2014; Modan etal., Clin. Exp. Dermatol. 34(5) 561-565, 2009; and Shi et al., J. Rheum44 (7) doi 10.3899/jrheum.161480).

Significant progress has been made in elucidating the role ofextracellular HARS derived proteins, including the identification of aputative cellular receptor, neuropilin-2 (NRP2 or NRP-2). Interactionsof HARS with NRP2 appear to be mediated by the N-terminal region ofHARS, and can lead to important changes in the cellular function ofNRP2.

Accordingly, the current discovery of the Resokine/neuropilin-2 axisrepresents a previously unknown mechanism, which acts as a centralregulator of cellular processes, including, for example, axonalguidance, endocytosis, cell migration, proliferation, survival,apoptosis, lymphangiogenesis, cellular differentiation, and cellattachment with direct relevance to cancer initiation, growth andmetastasis, as wells as muscular, vascular, neuronal, bone, and immunehomeostasis. The deregulation of any of these processes may lead to aspectrum of diseases, which may be addressed by the development ofanti-NRP2 antibodies that selectively target the Resokine/neuropilin-2axis. The present disclosure provides such antibodies and relatedembodiments.

BRIEF SUMMARY

Embodiments of the present disclosure include therapeutic compositions,comprising at least one antibody or antigen-binding fragment thereofthat specifically binds to a human neuropilin-2 (NRP2) polypeptide(anti-NRP2 antibody).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to a full-length human NRP2polypeptide or a human NRP2 polypeptide selected from Table N1,optionally with an affinity of about 10 pM to about 500 pM or to about50 nM, or about, at least about, or no more than about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM,or optionally with an affinity that ranges from about 10 pM to about 500pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM,about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM toabout 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM,or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pMto about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM toabout 50 nM. In some instances, the at least one antibody orantigen-binding fragment thereof specifically binds to the human NRP2polypeptide in its native form but does not substantially bind to thehuman NRP2 polypeptide in its denatured form.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof that specifically binds to at least one epitope in aneuropilin domain selected from one or more of the neuropilin A1 domain,neuropilin A2 domain, neuropilin B1 domain, neuropilin B2 domain,neuropilin C domain, neuropilin A1/A2 combined domain, neuropilin B1/B2combined domain, neuropilin A2/B1 combined domain, neuropilin B2/Ccombined domain, neuropilin A2/B1/B2 combined domain, neuropilinA2/B1/B2/C combined domain, neuropilin A1/A2/B1 combined domain,neuropilin A1/A2/B1/B2 combined domain, neuropilin A1/A2/B1/B2/Ccombined domain, and the neuropilin B1/B2/C combined domain, optionallywith an affinity of about 10 pM to about 500 pM or to about 50 nM, orabout, at least about, or no more than about 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400,500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or optionallywith an affinity that ranges from about 10 pM to about 500 pM, about 10pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, orabout 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM toabout 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM,about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pMto about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nMto about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin A1 domain, the neuropilin A2 domain, and/or the neuropilinA1A2 combined domain, including adjacent linker regions, for example, atabout residues;

(neuropilin A1 domain) 20-148, 30-141, 40-141, 50-141, 60-141, 70-141,80-141, 90-141, 100-141, 110-141, 120-141, 130-141; 20-130, 20-120,20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 asdefined by SEQ ID NO:1 (FL human NRP2); or

(neuropilin A2 domain) 142-280, 150-265, 160-265, 170-265, 180-265,190-265, 200-265, 210-265, 220-265, 230-265, 240-265, 250-265, 260-265,141-270, 141-260, 141-250, 141-240, 141-230, 141-220, 141-210, 141-200,141-190, 141-180, 141-170, 141-160, 141-150, 200-250, 210-250, 220-250,230-250, 200-240, 210-240, 220-240, 230-240, 227-247, 228-247, 229-247,230-247, 231-247, 232-247, 233-247, 234-247, 235-247, 236-247; 227-246,227-245, 227-244, 227-243, 227-242, 227-241, 227-240, 227-239, 227-238;235-240, 236-239, 236-238, or residue 237 as defined by SEQ ID NO:1 (FLhuman NRP2); or

(combined A1A2 domain) 20-280, 30-280, 40-280, 50-280, 60-280, 70-280,80-280, 90-280, 100-280, 110-280, 120-280, 130-280, 140-280, 150-280,160-280, 170-280, 180-280, 190-280, 200-280, 210-280, 220-280, 230-280,240-280, 260-280, 270-280, 20-270, 20-260, 20-250, 20-240, 20-230,20-220, 20-210, 20-200, 20-190, 20-180, 20-170, 20-160, 20-150, 20-140,20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50,20-40, or 20-30 as defined by SEQ ID NO:1 (FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin B1 domain (SEQ ID NO:12), the neuropilin B2 domain (SEQ IDNO:13), and/or the neuropilin B1/B2 combined domain (SEQ ID NO:20),including adjacent linker regions; for example, at about residues;

(neuropilin B1 domain) 266-426, 280-426, 290-426, 300-426, 310-426,320-426, 330-426, 340-426, 350-426, 360-426, 370-426, 380-426, 390-426,400-426, 410-426, 420-426, 280-420, 280-410, 280-400, 280-390, 280-380,280-370, 280-360, 280-350, 280-340, 280-330, 280-320, 280-310, 280-300,or 280-290 as defined by SEQ ID NO:1 (FL human NRP2);

(neuropilin B2 domain) 438-591, 450-591, 460-591, 470-591, 480-591,490-591, 500-591, 510-591, 520-591, 530-591, 540-591, 550-591, 560-591,570-591, 580-591, 438-590, 438-580, 438-570, 438-560, 438-550, 438-540,438-530, 438-520, 438-510, 438-500, 438-490, 438-480, 438-470, 438-460,or 438-450 as defined by SEQ ID NO:1 (FL human NRP2); or

(neuropilin B1/B2 combined domain) 266-591, 276-591, 286-591, 296-591,306-591, 316-591, 326-591, 336-591, 346-591, 356-591, 366-591, 376-591,386-591, 396-591, 406-591, 416-591, 426-591, 436-591, 446-591, 456-591,466-591, 476-591, 486-591, 498-591, 508-591, 518-591, 528-591, 538-591,548-591, 558-591, 568-591, 578-591, 588-591, 266-581, 266-571, 266-561,266-551, 266-541, 266-531, 266-521, 266-511, 266-501, 266-491, 266-481,266-471, 266-461, 266-451, 266-441, 266-431, 266-421, 266-411, 266-401,266-391, 266-381, 266-371, 266-361, 266-351, 266-341, 266-331, 266-321,266-311, 266-301, 266-291, 266-281, or 266-271 as defined by SEQ ID NO:1(FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin A2/B1 combined domain and/or the neuropilin B2C combineddomain, including adjacent linker regions, for example, at aboutresidues;

(neuropilin A2B1 combined domain) 149-437, 159-426, 169-426, 179-426,189-426, 199-426, 209-426, 219-426, 229-426,239-426, 249-426, 259-426,269-426, 279-426, 289-426, 299-426, 309-426, 319-426, 329-426, 339-426,349-426, 359-426, 369-426, 379-426, 389-426, 399-426, 409-426, 419-426,149-436, 149-426, 149-416, 149-406, 149-396, 149-386, 149-376, 149-366,149-356, 149-346, 149-336, 149-326, 149-316, 149-306, 149-296, 149-286,149-276, 149-266, 149-256, 149-246, 149-236, 149-226, 149-216, 149-206,149-196, 146-186, 146-176, 146-166, or 146-155 as defined by SEQ ID NO:1(FL human NRP2); or

(neuropilin B2C combined domain) 438-794, 448-794, 458-794, 468-794,478-794, 487-794, 497-794, 507-794, 517-794, 527-794, 537-794, 547-794,557-794, 567-794, 587-794, 597-794, 607-794, 617-794, 627-794, 637-794,647-794, 657-794, 667-794, 677-794, 687-794, 697-794, 707-794, 717-794,727-794, 737-794, 747-794, 757-794, 767-794, 777-794, 787-794, 427-794,438-784, 438-774, 438-764, 438-754, 438-744, 438-734, 438-728, 438-714,438-704, 438-694, 438-684, 438-674, 438-664, 438-654, 438-644, 438-634,438-624, 438-614, 438-604, 438-596, 438-586, 438-576, 438-566, 438-556,438-546, 438-536, 438-526, 438-516, 438-506, 438-494, 438-484, 438-474,438-464, 438-454, 438-444 as defined by SEQ ID NO:1 (FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin C domain, including adjacent linker regions, for example, atabout residues 591-794, 600-794, 610-794, 620-794, 630-794, 640-794,650-794, 660-794, 670-794, 680-794, 690-794, 700-794, 710-794, 720-794,730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 591-790,591-780, 591-770, 591-760, 591-750, 591-740, 591-730, 591-720, 591-710,591-700, 591-690, 591-680, 591-670, 591-660, 591-650, 591-640, 591-630,591-620, 591-610, or 591-600 as defined by SEQ ID NO:1 (FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin B1/B2/C combined domain, including adjacent linker regions,for example, at about residues 276-794, 286-794, 296-794, 306-794,316-794, 326-794, 336-794, 346-794, 356-794, 366-794, 376-794, 387-794,396-794, 406-794, 416-794, 426-794, 436-794, 446-794, 456-794, 466-794,476-794, 486-794, 496-794, 506-794, 516-794, 526-794, 536-794, 546-794,556-794, 566-794, 576-794, 586-794, 596-794, 606-794, 616-794, 626-794,636-794, 646-794, 656-794, 666-794, 676-794, 686-794, 696-794, 706-794,716-794, 726-794, 736-794, 746-794, 756-794, 766-794, 776-794, 786-794,266-794, 276-784, 276-774, 276-764, 276-754, 276-744, 276-734, 276-724,276-714, 276-704, 276-694, 276-684, 276-674, 276-664, 276-654, 276-644,276-634, 276-624, 276-614, 276-604, 276-594, 276-584, 276-574, 276-564,276-554, 276-544, 276-534, 276-524, 276-514, 276-504, 276-594, 276-584,276-574, 276-564, 276-554, 276-544, 276-534, 276-524, 276-514, 276-504,or 276-496 as defined by SEQ ID NO:1 (FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to a conformational epitope composedof two or more discontinuous epitope regions. In some embodiments, theat least one antibody or antigen-binding fragment thereof specificallybinds to a conformational epitope comprising or consisting of:

(a) a first epitope region within the A1 domain, and second epitoperegion within the A2 domain of the human NPR2 polypeptide;

(b) a first epitope region within the A1 domain, and second epitoperegion within the B1 domain of the human NPR2 polypeptide;

(c) a first epitope region within the A1 domain, and second epitoperegion within the B2 domain of the human NPR2 polypeptide;

(d) a first epitope region within the A1 domain, and second epitoperegion within the C domain of the human NPR2 polypeptide;

(e) a first epitope region within the A2 domain, and second epitoperegion within the B1 domain of the human NPR2 polypeptide;

(f) a first epitope region within the A2 domain, and second epitoperegion within the B2 domain of the human NPR2 polypeptide;

(g) a first epitope region within the A2 domain, and second epitoperegion within the C domain of the human NPR2 polypeptide;

(h) a first epitope region within the B1 domain, and second epitoperegion within the B2 domain of the human NPR2 polypeptide;

(i) a first epitope region within the B1 domain, and second epitoperegion within the C domain of the human NPR2 polypeptide; or

(j) a first epitope region within the B2 domain, and second epitoperegion within the C domain of the human NPR2 polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof modulates (e.g., interferes with) binding of the humanNRP2 polypeptide to at least one NRP2 ligand (for example, an NRP2ligand selected from Table N2 or Table N3 and/or a human histidyl-tRNAsynthetase (HRS) polypeptide selected from Table H1). In someembodiments, the at least NRP2 ligand is an HRS splice variant selectedfrom Table H1, for example, a HRS splice variant selected from one ormore of SV9 (HRS(1-60)), SV11 (HRS(1-60)+(399-509)) and SV14(HRS(1-100)+(399-509)).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof is a blocking antibody which inhibits about or at leastabout 80-100% of the theoretical maximal binding between the human NRP2polypeptide and the at least one NRP2 ligand after pre-incubation withthe human NRP2 polypeptide in a stoichiometrically equivalent amount,optionally about or at least about 80, 85, 90, 95, or 100% of thetheoretical maximal binding.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof is a partial blocking antibody which inhibits about orat least about 20-80% of the theoretical maximal binding between thehuman NRP2 polypeptide and the at least one NRP2 ligand afterpre-incubation with the human NRP2 polypeptide in a stoichiometricallyequivalent amount, optionally about or at least about 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, or 80% of the theoretical maximalbinding.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an HRS polypeptide-interactingregion of the NRP2 polypeptide, and mimics or agonizes one or moresignaling activities of the HRS polypeptide binding to the NRP2polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an HRS polypeptide-interactingregion of the NRP2 polypeptide, and modulates binding/signaling activitybetween the NRP2 polypeptide and at least one NRP2 ligand. In someembodiments, the at least one antibody or antigen-binding fragmentthereof antagonizes the binding/signaling activity between the NRP2polypeptide and the at least one NRP2 ligand. In some embodiments, theat least one antibody or antigen-binding fragment thereof agonizes orenhances the binding/signaling activity between the NRP2 polypeptide andthe at least one NRP2 ligand.

In some embodiments, the at least one NRP2 ligand is selected from oneor more of:

-   -   a VEGF selected from one or more of VEGF-A145, VEGF-A165,        VEGF-C, VEGF-D and PIGF-2;    -   a VEGF receptor (VEGFR) selected from VEGFR2 and VEGFR3;    -   a semaphorin selected from one or more of SEMA-3B, SEMA-3C,        SEMA-3D SEMA-3F, and SEMA-3G;    -   a plexin selected from one or more of plexin A1, A2, A3, A4, and        D1;    -   a growth factor selected from one or more of fibroblast growth        factor (FGF), hepatocyte growth factor (HGF), and platelet        derived growth factor (PDGF);    -   a growth factor receptor selected from one or more of a        fibroblast growth factor receptor (FGFR), a hepatocyte growth        factor receptor (HGFR), and a platelet derived growth factor        receptor (PDGF);    -   a galectin or a galectin receptor    -   a transcription factor selected from FAC1 and bromoprotein PHD        finger transcription factor;    -   an adaptor protein selected from one or more of GIPC1, GIPC2 and        GIPC3;    -   an integrin selected from Table N3, optionally one or more of        α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈, α6β₁ and α₆β₄;    -   a transforming growth factor beta selected from one or more of        TGFβ1, TGFβ₂, TGFβ₃, and their corresponding TGFβ receptors; and    -   an HRS polypeptide selected from Table H1, optionally an HRS        splice variant selected from one or more of HisRS^(N1),        HisRS^(N2), HisRS^(N3), HisRS^(N4)(SV9), HisRS^(N5), HisRS^(C1),        HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5), HisRS^(C6),        HisRS^(C7), HisRS^(C8)(SV11), and HisRS^(C9) (SV14).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or a semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and VEGFR3 or VEGF-C.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and a HRS polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or a semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and a HRS polypeptide, and without substantially modulatingthe binding/signaling activity between the NRP2 polypeptide and VEGFR3or VEGF-C.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and VEGR3 without substantially modulating thebinding/signaling activity between the NRP2 polypeptide and a plexinreceptor and/or a semaphorin.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and VEGR3 or VEGF-C without substantially modulatingthe binding/signaling activity between the NRP2 polypeptide and a HRSpolypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor without substantially modulatingthe ligand binding of semaphorin 3 to NRP2.

In some embodiments, the plexin receptor is selected from plexin A1, A2,A3, A4, and D1. In some embodiments, the semaphorin is selected fromsemaphorin 3B, 3C, 3D, 3F, and 3G.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope within the human NRP2A2 domain which comprises at least 5 contiguous amino acids of SEQ IDNO: 11, wherein the at least one antibody or antigen-binding fragmentthereof selectively inhibits receptor dimerization between NRP2 andplexin A1 without substantially inhibiting dimerization between NRP2 andFLT4 (VEGFR3). In some embodiments, the at least one antibody orantigen-binding fragment thereof specifically binds to an epitope withinamino acids 232-242 of human NRP2 SEQ ID NO: 1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope within the human NRP2B1 domain which comprises at least 5 contiguous amino acids of SEQ IDNO: 12, wherein the at least one antibody or antigen-binding fragmentthereof selectively inhibits receptor dimerization between NRP2 and FLT4(VEGFR3) without substantially inhibiting dimerization between NRP2 andplexin A1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope within the human NRP2B2 domain which comprises at least 5 contiguous amino acids of SEQ IDNO: 13, wherein the at least one antibody or antigen-binding fragmentthereof inhibits receptor dimerization between NRP2 and FLT4 (VEGFR3)and inhibits dimerization between NRP2 and plexin A1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope within the human NRP2C domain which comprises at least 5 contiguous amino acids of SEQ ID NO:14, wherein the at least one antibody or antigen-binding fragmentthereof inhibits receptor dimerization between NRP2 and plexin A1 andpartially inhibits dimerization between NRP2 and FLT4 (VEGFR3).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof has an affinity (Kd or EC₅₀) for each of (i) a humanNRP2 polypeptide and (ii) the corresponding region of a cynomolgusmonkey NRP2 polypeptide, wherein the affinity for (i) and (ii) is withinthe range of about 20 pM to about 200 pM, about 30 pM to about 300 pM,about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM toabout 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM,about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 0.4 toabout 1.2 nM, about 0.9 to about 5.5 nM, about 0.9 to about 5 nM, orabout 1 nM to about 10 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof has an affinity (Kd or EC₅₀) for each of (i) a humanNRP2 polypeptide and (ii) the corresponding region of a murine NRP2polypeptide, wherein the affinity for (i) and (ii) is within the rangeof about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pMto about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90pM to about 900 pM, about 100 pM to about 1 nM, or about 1 nM to about10 nM.

In certain embodiments, the at least one antibody or antigen-bindingfragment thereof comprises:

a heavy chain variable region (V_(H)) sequence that comprisescomplementary determining region V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3sequences selected from Table A1 and variants thereof which specificallybind to the human NRP2 polypeptide; and

a light chain variable region (V_(L)) sequence that comprisescomplementary determining region V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences selected from Table A1 and variants thereof which specificallybind to the human NRP2 polypeptide,

including affinity matured variants of the foregoing which specificallybind to the human NRP2 polypeptide.

In specific embodiments:

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:23-25, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 26-28, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:29-31, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 32-34, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:35-37, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 38-40, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:41-43, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 44-46, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:47-49, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 50-52, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:53-55, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 56-58, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:59-61, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 62-64, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:65-67, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 68-70, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:71-73, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 74-76, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:77-79, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 80-82, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:83-85, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 86-88, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof comprises an IgA (including subclasses IgA1 and IgA2),IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), or IgMFc domain, optionally a human Fc domain, or a hybrid and/or variantthereof. In some embodiments, the at least one antibody orantigen-binding fragment thereof comprises an IgG Fc domain with higheffector function in humans, optionally an IgG1 or IgG3 Fc domain. Insome embodiments, the at least one antibody or antigen-binding fragmentthereof comprises an IgG Fc domain with low effector function in humans,optionally an IgG2 or IgG4 Fc domain. In some embodiments, the at leastone antibody or antigen-binding fragment thereof comprises an IgG1 orIgG4 Fc domain, optionally selected from Table F1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof is a monoclonal antibody. In some embodiments, the atleast one antibody or antigen-binding fragment thereof is a humanizedantibody. In some embodiments, the at least one antibody orantigen-binding fragment thereof is an Fv fragment, a single chain Fv(scFv) polypeptide, an adnectin, an anticalin, an aptamer, an avimer, acamelid antibody, a designed ankyrin repeat protein (DARPin), aminibody, a nanobody, or a unibody.

In some embodiments, the composition has a purity of at least about 80%,85%, 90%, 95%, 98%, or 99% on a protein basis with respect to the atleast one antibody or antigen-binding fragment, and is substantiallyaggregate-free. In some embodiments, the therapeutic composition issubstantially endotoxin-free.

In some embodiments, the therapeutic composition is a sterile,injectable solution, optionally suitable for intravenous, intramuscular,subcutaneous, or intraperitoneal administration.

In certain embodiments, the therapeutic composition further comprises atleast one additional agent selected from one or more of a cancerimmunotherapy agent, a chemotherapeutic agent, a hormonal therapeuticagent, and a kinase inhibitor. In some embodiments, the cancerimmunotherapy agent is selected from one or more of an immune checkpointmodulatory agent, a cancer vaccine, an oncolytic virus, a cytokine, anda cell-based immunotherapies. In some embodiments, the immune checkpointmodulatory agent is a polypeptide, optionally an antibody orantigen-binding fragment thereof or a ligand, or a small molecule. Insome embodiments, the immune checkpoint modulatory agent comprises

(a) an antagonist of a inhibitory immune checkpoint molecule; or

(b) an agonist of a stimulatory immune checkpoint molecule,

for instance, wherein the immune checkpoint modulatory agentspecifically binds to the immune checkpoint molecule.

In some embodiments, the inhibitory immune checkpoint molecule isselected from one or more of Programmed Death-Ligand 1 (PD-L1),Programmed Death 1 (PD-1), Programmed Death-Ligand 2 (PD-L2), CytotoxicT-Lymphocyte-Associated protein 4 (CTLA-4), Indoleamine 2,3-dioxygenase(IDO), tryptophan 2,3-dioxygenase (TDO), T-cell Immunoglobulin domainand Mucin domain 3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3),V-domain Ig suppressor of T cell activation (VISTA), B and T LymphocyteAttenuator (BTLA), CD160, Herpes Virus Entry Mediator (HVEM), and T-cellimmunoreceptor with Ig and ITIM domains (TIGIT).

In some embodiments, the antagonist is a PD-L1 and/or PD-L2 antagonistoptionally selected from one or more of an antibody or antigen-bindingfragment or small molecule that specifically binds thereto, atezolizumab(MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736);

the antagonist is a PD-1 antagonist optionally selected from one or moreof an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP-224,AMP-514, PDR001, and pidilizumab;

the antagonist is a CTLA-4 antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, ipilimumab, and tremelimumab;

the antagonist is an IDO antagonist optionally selected from one or moreof an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, indoximod (NLG-8189), 1-methyl-tryptophan(1MT), β-Carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinicacid, and epacadostat;

the antagonist is a TDO antagonist optionally selected from one or moreof an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, 680C91, and LM10;

the antagonist is a TIM-3 antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto;

the antagonist is a LAG-3 antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, and BMS-986016;

the antagonist is a VISTA antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto;

the antagonist is a BTLA, CD160, and/or HVEM antagonist optionallyselected from one or more of an antibody or antigen-binding fragment orsmall molecule that specifically binds thereto;

and/or the antagonist is a TIGIT antagonist optionally selected from oneor more of an antibody or antigen-binding fragment or small moleculethat specifically binds thereto.

In some embodiments, the stimulatory immune checkpoint molecule isselected from one or more of OX40, CD40, Glucocorticoid-Induced TNFRFamily Related Gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and HerpesVirus Entry Mediator (HVEM).

In some embodiments, the agonist is an OX40 agonist optionally selectedfrom one or more of an antibody or antigen-binding fragment or smallmolecule or ligand that specifically binds thereto, OX86, Fc-OX40L, andGSK3174998;

the agonist is a CD40 agonist optionally selected from one or more of anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, CP-870,893, dacetuzumab, Chi Lob 7/4,ADC-1013, and rhCD40L;

the agonist is a GITR agonist optionally selected from one or more of anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, INCAGN01876, DTA-1, and MEDI1873;

the agonist is a CD137 agonist optionally selected from one or more ofan antibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, utomilumab, and 4-1BB ligand;

the agonist is a CD27 agonist optionally selected from one or more of anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, varlilumab, and CDX-1127 (1F5);

the agonist is a CD28 agonist optionally selected from one or more of anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, and TAB08; and/or

the agonist is an HVEM agonist optionally selected from one or more ofan antibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto.

In some embodiments, the cancer vaccine is selected from one or more ofOncophage, a human papillomavirus HPV vaccine optionally Gardasil orCervarix, a hepatitis B vaccine optionally Engerix-B, Recombivax HB, orTwinrix, and sipuleucel-T (Provenge), or comprises a cancer antigenselected from one or more of human Her2/neu, HerVEGF receptor (EGFR),Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE Receptor),MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growthfactor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40,CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR,CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1(IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA),guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrinα5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblastactivation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (orCA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA),NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamilymember 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin.

In some embodiments, the oncolytic virus selected from one or more oftalimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™),Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010),Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102(Ad5/3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401. In some embodiments,the cytokine selected from one or more of interferon (IFN)-α, IL-2,IL-12, IL-7, IL-21, and Granulocyte-macrophage colony-stimulating factor(GM-CSF). In some embodiments, the cell-based immunotherapy agentcomprises cancer antigen-specific T-cells, optionally ex vivo-derivedT-cells. In some embodiments, the cancer antigen-specific T-cells areselected from one or more of chimeric antigen receptor (CAR)-modifiedT-cells, and T-cell Receptor (TCR)-modified T-cells, tumor infiltratinglymphocytes (TILs), and peptide-induced T-cells.

In some embodiments, the at least one chemotherapeutic agent is selectedfrom one or more of an alkylating agent, an anti-metabolite, a cytotoxicantibiotic, a topoisomerase inhibitor (type 1 or type II), and ananti-microtubule agent.

In some embodiments, the alkylating agent is selected from one or moreof nitrogen mustards (optionally mechlorethamine, cyclophosphamide,mustine, melphalan, chlorambucil, ifosfamide, and busulfan),nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine(BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, andstreptozotocin), tetrazines (optionally dacarbazine, mitozolomide, andtemozolomide), aziridines (optionally thiotepa, mytomycin, anddiaziquone (AZQ)), cisplatins and derivatives thereof (optionallycarboplatin and oxaliplatin), and non-classical alkylating agents(optionally procarbazine and hexamethylmelamine);

the anti-metabolite is selected from one or more of anti-folates(optionally methotrexate and pemetrexed), fluoropyrimidines (optionally5-fluorouracil and capecitabine), deoxynucleoside analogues (optionallyancitabine, enocitabine, cytarabine, gemcitabine, decitabine,azacitidine, fludarabine, nelarabine, cladribine, clofarabine,fludarabine, and pentostatin), and thiopurines (optionally thioguanineand mercaptopurine);

the cytotoxic antibiotic is selected from one or more of anthracyclines(optionally doxorubicin, daunorubicin, epirubicin, idarubicin,pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C,mitoxantrone, and actinomycin;

the topoisomerase inhibitor is selected from one or more ofcamptothecin, irinotecan, topotecan, etoposide, doxorubicin,mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; and/or

the anti-microtubule agent is selected from one or more of taxanes(optionally paclitaxel and docetaxel) and vinca alkaloids (optionallyvinblastine, vincristine, vindesine, vinorelbine).

In some embodiments, the at least one hormonal therapeutic agent is ahormonal agonist or a hormonal antagonist. In some embodiments, thehormonal agonist is selected from one or more of a progestogen(progestin), a corticosteroid (optionally prednisolone,methylprednisolone, or dexamethasone), insulin like growth factors, VEGFderived angiogenic and lymphangiogenic factors (optionally VEGF-A,VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor(FGF), galectin, hepatocyte growth factor (HGF), platelet derived growthfactor (PDGF), transforming growth factor (TGF)-beta, an androgen, anestrogen, and a somatostatin analog. In some embodiments, the hormonalantagonist is selected from one or more of a hormone synthesisinhibitor, optionally an aromatase inhibitor or a gonadotropin-releasinghormone (GnRH) or an analog thereof, and a hormone receptor antagonist,optionally a selective estrogen receptor modulator (SERM) or ananti-androgen, or an antibody directed against a hormonal receptor,optionally cixutumumab, dalotuzumab, figitumumab, ganitumab,istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab,ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab,depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine,matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab,tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab,olaratumab, or tovetumab.

In some embodiments, the kinase inhibitor is selected from one or moreof adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib,cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib,entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib,imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab,pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib,sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, andvemuafenib.

Also included are methods of treating a disease or condition in asubject in need thereof, comprising administering to the subject atherapeutic composition comprising at least one antibody orantigen-binding fragment thereof that specifically binds to a humanneuropilin-2 (NRP2) polypeptide, wherein the at least one antibody orantigen-binding fragment thereof modulates (e.g., interferes with)binding of the human NRP2 polypeptide to at least one NRP2 ligand (forexample, an NRP2 ligand from Table N2 or Table N3 and/or a humanhistidyl-tRNA synthetase (HRS) polypeptide from Table H1), for example,as a therapeutic composition described herein.

In some embodiments, the disease or condition is an NRP2-associateddisease or condition. In some embodiments, the NRP2-associated diseaseor condition is selected from one or more of cancer and diseases andpathways associated with cancer, including cancer cell growth,initiation, migration, adhesion, invasion, and/or metastasis; diseasesassociated with inflammation, autoimmunity, and related inflammatorydiseases, including diseases associated with inappropriate immune cellactivation or migration such as Graft versus host disease (GVHD);diseases associated with lymphatic development, lymphangiogenesis, andlymphatic damage, including, for example, edema, lymphedema, secondarylymphedema, inappropriate fat absorption and deposition, excess fatdeposition, and vascular permeability; diseases associated withinfections, including latent infections; diseases associated withallergic disorders/diseases, allergic responses, including, for example,chronic obstructive pulmonary disorder (COPD), neutrophilic asthma,antineutrophil cytoplasmic antibody (ANCA)-associated systemicvasculitis, systemic lupus erythematosus, rheumatoid arthritis,inflammasome-related diseases, and skin-related neutrophil-mediateddiseases such as pyoderma gangrenosum; diseases associated withgranulomatous inflammatory diseases, including sarcoidosis andgranulomas; diseases associated with fibrosis including fibroticdiseases, fibrosis, endothelial to mesenchymal transition (EMT), andwound healing; diseases associated with inappropriate smooth musclecontractility, and inappropriate vascular smooth muscle cell migrationand adhesion; diseases associated with inappropriate autophagy,phagocytosis, and efferocytosis; diseases associated with inappropriatemigratory cell movement; diseases associated with neuronal diseases,peripheral nervous system remodeling, and pain perception; and diseasesassociated with bone development and bone remodeling.

In some embodiments, the disease is a cancer, for example, wherein thecancer expresses or overexpresses NRP2. In some embodiments, the cancerdisplays NRP2-dependent growth, NRP2-dependent adhesion, NRP2-dependentmigration, and/or NRP2-dependent invasion. In some embodiments thecancer expresses or overexpresses NRP2 but does not substantiallyexpress neuropilin-1 (NRP1).

Also included are methods for reducing or preventing re-emergence of acancer in a subject in need thereof, wherein administration of thetherapeutic composition enables generation of an immune memory to thecancer. In some embodiments, the subject has or is at risk fordeveloping diabetes.

Certain methods comprise administering to the subject at least oneadditional agent selected from one or more of a cancer immunotherapyagent, a chemotherapeutic agent, a hormonal therapeutic agent, and akinase inhibitor, for example, as described herein. In some embodiments,the at least one anti-NRP2 antibody or antigen-binding fragment thereofand the at least one agent are administered separately, as separatecompositions. In some embodiments, the at least one anti-NRP2 antibodyand the at least one agent are administered together as part of the sametherapeutic composition, for instance, as a therapeutic compositiondescribed herein.

In some embodiments, the cancer immunotherapy agent is selected from oneor more of an immune checkpoint modulatory agent, a cancer vaccine, anoncolytic virus, a cytokine, and a cell-based immunotherapies. In someembodiments, the immune checkpoint modulatory agent is a polypeptide,optionally an antibody or antigen-binding fragment thereof or a ligand,or a small molecule. In some embodiments, the immune checkpointmodulatory agent comprises

(a) an antagonist of a inhibitory immune checkpoint molecule; or

(b) an agonist of a stimulatory immune checkpoint molecule.

for example, wherein the immune checkpoint modulatory agent specificallybinds to the In some embodiments, the inhibitory immune checkpointmolecule is selected from one or more of Programmed Death-Ligand 1(PD-L1), Programmed Death 1 (PD-1), Programmed Death-Ligand 2 (PD-L2),Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), Indoleamine2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cellImmunoglobulin domain and Mucin domain 3 (TIM-3), Lymphocyte ActivationGene-3 (LAG-3), V-domain Ig suppressor of T cell activation (VISTA), Band T Lymphocyte Attenuator (BTLA), CD160, Herpes Virus Entry Mediator(HVEM), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).

In some embodiments, the antagonist is a PD-L1 and/or PD-L2 antagonistoptionally selected from one or more of an antibody or antigen-bindingfragment or small molecule that specifically binds thereto, atezolizumab(MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736),optionally wherein the cancer is selected from one or more of colorectalcancer, melanoma, breast cancer, non-small-cell lung carcinoma, bladdercancer, and renal cell carcinoma;

the antagonist is a PD-1 antagonist optionally selected from one or moreof an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP-224,AMP-514PDR001, and pidilizumab, optionally wherein the PD-1 antagonistis nivolumab and the cancer is optionally selected from one or more ofHodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellularcarcinoma, renal cell carcinoma, and ovarian cancer;

the PD-1 antagonist is pembrolizumab and the cancer is optionallyselected from one or more of melanoma, non-small cell lung cancer, smallcell lung cancer, head and neck cancer, and urothelial cancer;

the antagonist is a CTLA-4 antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, ipilimumab, tremelimumab, optionally whereinthe cancer is selected from one or more of melanoma, prostate cancer,lung cancer, and bladder cancer;

the antagonist is an IDO antagonist optionally selected from one or moreof an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, indoximod (NLG-8189), 1-methyl-tryptophan(1MT), β-Carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinicacid, and epacadostat, and wherein the cancer is optionally selectedfrom one or more of metastatic breast cancer and brain cancer optionallyglioblastoma multiforme, glioma, gliosarcoma or malignant brain tumor;

the antagonist is a TDO antagonist optionally selected from one or moreof an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, 680C91, and LM10;

the antagonist is a TIM-3 antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto;

the antagonist is a LAG-3 antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto, and BMS-986016;

the antagonist is a VISTA antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto;

the antagonist is a BTLA, CD160, and/or HVEM antagonist optionallyselected from one or more of an antibody or antigen-binding fragment orsmall molecule that specifically binds thereto;

the antagonist is a TIGIT antagonist optionally selected from one ormore of an antibody or antigen-binding fragment or small molecule thatspecifically binds thereto.

In some embodiments, the stimulatory immune checkpoint molecule isselected from one or more of OX40, CD40, Glucocorticoid-Induced TNFRFamily Related Gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and HerpesVirus Entry Mediator (HVEM).

In some embodiments, the agonist is an OX40 agonist optionally selectedfrom one or more of an antibody or antigen-binding fragment or smallmolecule or ligand that specifically binds thereto, OX86, Fc-OX40L, andGSK3174998;

the agonist is a CD40 agonist optionally selected from one or more of anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, CP-870,893, dacetuzumab, Chi Lob 7/4,ADC-1013, and rhCD40L, and wherein the cancer is optionally selectedfrom one or more of melanoma, pancreatic carcinoma, mesothelioma, andhematological cancers optionally lymphoma such as Non-Hodgkin'slymphoma;

the agonist is a GITR agonist optionally selected from one or more of anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, INCAGN01876, DTA-1, and MEDI1873;

the agonist is a CD137 agonist optionally selected from one or more ofan antibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, utomilumab, and 4-1BB ligand;

the agonist is a CD27 agonist optionally selected from one or more of anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, varlilumab, and CDX-1127 (1F5);

the agonist is a CD28 agonist optionally selected from one or more of anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto, and TAB08; and/or

the agonist is an HVEM agonist optionally selected from one or more ofan antibody or antigen-binding fragment or small molecule or ligand thatspecifically binds thereto.

In some embodiments, the cancer vaccine is selected from one or more ofOncophage, a human papillomavirus HPV vaccine optionally Gardasil orCervarix, a hepatitis B vaccine optionally Engerix-B, Recombivax HB, orTwinrix, and sipuleucel-T (Provenge), or comprises a cancer antigenselected from one or more of human Her2/neu, HerVEGF receptor (EGFR),Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE Receptor),MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growthfactor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40,CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR,CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1(IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA),guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrinα5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblastactivation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (orCA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA),NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamilymember 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin, optionally wherein the subject has oris at risk for having a cancer that comprises the corresponding cancerantigen.

In some embodiments, the oncolytic virus selected from one or more oftalimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™),Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010),Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102(Ad5/3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401. In some embodiments,the cytokine selected from one or more of interferon (IFN)-α, IL-2,IL-12, IL-7, IL-21, and Granulocyte-macrophage colony-stimulating factor(GM-CSF). In some embodiments, the cell-based immunotherapy agentcomprises cancer antigen-specific T-cells, optionally ex vivo-derivedT-cells. In some embodiments, the cancer antigen-specific T-cells areselected from one or more of chimeric antigen receptor (CAR)-modifiedT-cells, and T-cell Receptor (TCR)-modified T-cells, tumor infiltratinglymphocytes (TILs), and peptide-induced T-cells.

In some embodiments, the at least one chemotherapeutic agent is selectedfrom one or more of an alkylating agent, an anti-metabolite, a cytotoxicantibiotic, a topoisomerase inhibitor (type 1 or type II), and ananti-microtubule agent.

In some embodiments, the alkylating agent is selected from one or moreof nitrogen mustards (optionally mechlorethamine, cyclophosphamide,mustine, melphalan, chlorambucil, ifosfamide, and busulfan),nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine(BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, andstreptozotocin), tetrazines (optionally dacarbazine, mitozolomide, andtemozolomide), aziridines (optionally thiotepa, mytomycin, anddiaziquone (AZQ)), cisplatins and derivatives thereof (optionallycarboplatin and oxaliplatin), and non-classical alkylating agents(optionally procarbazine and hexamethylmelamine);

the anti-metabolite is selected from one or more of anti-folates(optionally methotrexate and pemetrexed), fluoropyrimidines (optionally5-fluorouracil and capecitabine), deoxynucleoside analogues (optionallyancitabine, enocitabine, cytarabine, gemcitabine, decitabine,azacitidine, fludarabine, nelarabine, cladribine, clofarabine,fludarabine, and pentostatin), and thiopurines (optionally thioguanineand mercaptopurine);

the cytotoxic antibiotic is selected from one or more of anthracyclines(optionally doxorubicin, daunorubicin, epirubicin, idarubicin,pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C,mitoxantrone, and actinomycin;

the topoisomerase inhibitor is selected from one or more ofcamptothecin, irinotecan, topotecan, etoposide, doxorubicin,mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; and/or

the anti-microtubule agent is selected from one or more of taxanes(optionally paclitaxel and docetaxel) and vinca alkaloids (optionallyvinblastine, vincristine, vindesine, vinorelbine).

In some embodiments, the at least one hormonal therapeutic agent is ahormonal agonist or a hormonal antagonist. In some embodiments, thehormonal agonist is selected from one or more of a progestogen(progestin), a corticosteroid (optionally prednisolone,methylprednisolone, or dexamethasone), insulin like growth factors, VEGFderived angiogenic and lymphangiogenic factors (optionally VEGF-A,VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor(FGF), galectin, hepatocyte growth factor (HGF), platelet derived growthfactor (PDGF), transforming growth factor (TGF)-beta, an androgen, anestrogen, and a somatostatin analog. In some embodiments, the hormonalantagonist is selected from one or more of a hormone synthesisinhibitor, optionally an aromatase inhibitor or a gonadotropin-releasinghormone (GnRH) or an analog thereof, and a hormone receptor antagonist,optionally a selective estrogen receptor modulator (SERM) or ananti-androgen, or an antibody directed against a hormonal receptor,optionally cixutumumab, dalotuzumab, figitumumab, ganitumab,istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab,ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab,depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine,matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab,tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab,olaratumab, or tovetumab.

In some embodiments, the kinase inhibitor is selected from one or moreof adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib,cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib,entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib,imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab,pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib,sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, andvemuafenib. In some embodiments, the cancer is a primary cancer. In someembodiments, the cancer is a metastatic cancer, for example, ametastatic cancer that expresses NRP2 and/or NRP2B. In some embodiments,the cancer is selected from one or more of melanoma (e.g., metastaticmelanoma), pancreatic cancer, bone cancer, prostate cancer, small celllung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia(e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloidleukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma(hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer,ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme,meningioma, pituitary adenoma, vestibular schwannoma, primary CNSlymphoma, primitive neuroectodermal tumor (medulloblastoma), kidneycancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer,esophageal cancer, brain cancer, head and neck cancers, cervical cancer,testicular cancer, thyroid cancer, and stomach cancer.

In some embodiments, the metastatic cancer is selected from one or moreof:

(a) a bladder cancer which has metastasized to the bone, liver, and/orlungs;

(b) a breast cancer which has metastasized to the bone, brain, liver,and/or lungs;

(c) a colorectal cancer which has metastasized to the liver, lungs,and/or peritoneum;

(d) a kidney cancer which has metastasized to the adrenal glands, bone,brain, liver, and/or lungs;

(e) a lung cancer which has metastasized to the adrenal glands, bone,brain, liver, and/or other lung sites;

(f) a melanoma which has metastasized to the bone, brain, liver, lung,and/or skin/muscle;

(g) a ovarian cancer which has metastasized to the liver, lung, and/orperitoneum;

(h) a pancreatic cancer which has metastasized to the liver, lung,and/or peritoneum;

(i) a prostate cancer which has metastasized to the adrenal glands,bone, liver, and/or lungs;

(j) a stomach cancer which has metastasized to the liver, lung, and/orperitoneum;

(l) a thyroid cancer which has metastasized to the bone, liver, and/orlungs; and

(m) a uterine cancer which has metastasized to the bone, liver, lung,peritoneum, and/or vagina.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, increased circulating or serum levels of at least oneNRP2 ligand (for example, an NRP2 ligand from Table N2 or Table N3and/or an HRS polypeptide from Table H1), either bound or free, relativeto the levels of a healthy control or matched control standard orpopulation of subject(s), optionally about or at least about 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, or5000 pM of the at least one NRP2 ligand (for example, HRS polypeptide),or about or at least about 30-100, 40-100, 50-100, 30-2000, 40-2000,50-2000, 60-2000, 70-2000, 80-2000, 90-2000, 100-2000, 200-2000,300-2000, 400-2000, 500-2000, 600-2000, 700-2000, 800-2000, 900-2000,1000-2000, 2000-3000, 3000-4000, or 4000-5000 pM of the at least oneNRP2 ligand.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of at least one NRP2 ligand (for example, an NRP2 ligand fromTable N2 or Table N3 and/or an HRS polypeptide from Table H1) and/or acoding mRNA thereof relative to a healthy control or matched controlstandard or population of subject(s), optionally a cancer which hasincreased levels or expression of the at least one NRP2 ligand and/or acoding mRNA thereof relative to a non-cancerous control cell or tissue,optionally relative to a non-cancerous cell or tissue of the same typeas the cancer, optionally wherein the HRS polypeptide is a splicevariant selected from HisRS^(N1), HisRS^(N2), HisRS^(N3), HisRS^(N4),HisRS^(N5), HisRS^(C1), HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5),HisRS^(C6), HisRS^(C7), HisRS^(C8), and HisRS^(C9).

In some embodiments, the subject has, and/or is selected for treatmentbased on having, increased circulating or serum levels of a solubleneuropilin 2 (NRP2) polypeptide (for example, selected from Table N1),either bound or free, relative to the levels of a healthy control ormatched control standard or population of subject(s), optionallycirculating or serum levels of about or at least about 10, 20, 30, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000 pM of thesoluble NRP2 polypeptide, or optionally circulating or serum levelsabout 30-50, 50-100, 100-2000, 200-2000, 300-2000, 400-2000, 500-2000,600-2000, 700-2000, 800-2000, 900-2000, 1000-2000, 2000-3000, 3000-4000,4000-5000 pM of the soluble NRP2 polypeptide.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of an NRP2 polypeptide (for example, selected from Table N1)and/or a coding mRNA thereof relative to a healthy control or matchedcontrol standard or population of subject(s), optionally a cancer whichhas increased levels or expression of an NRP2 polypeptide (for example,selected from Table N1) and/or a coding mRNA thereof relative to acontrol cell or tissue, optionally relative to a non-cancerous cell ortissue of the same type as the cancer.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of NRP2A and/or NRP2B, or an altered ratio of NRP2A:NRP2Bexpression, relative to a healthy control or matched control standard orpopulation of subject(s). In some embodiments, the subject hassignificantly higher expression or levels of NRP2B relative to a healthycontrol or matched control standard or population of subject(s). In someembodiments, the levels of NRP2B are increased by about or at leastabout 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%,800%, 900%, 1000% compared to a healthy control or matched controlstandard or population of subject(s).

In some embodiments, the subject has, and/or is selected for treatmentbased on having, increased circulating levels of HRS:NRP2 complexesrelative to a healthy or matched control standard or population ofsubject(s).

In some embodiments, the healthy control or matched control standard orpopulation of subject(s) comprises average ranges for age-matchedsamples of cancerous or non-cancerous cells or tissue of the same typeas the cancer, which comprise specific characteristics such as drugresistance, metastatic potential, aggressiveness, genetic signature(e.g., p53 mutations, PTEN deletion, IGFR expression), and/or expressionpatterns.

Certain embodiments comprise administering the at least one anti-NRP2antibody in an amount and at a frequency sufficient to achieve anaverage, sustained serum or circulating levels of a soluble NRP2polypeptide of about or less than about 500 pM, 400 pM, 300 pM, 200 pM,100 pM, 50 pm, 40 pM, 30 pM, 20 pM, or 10 pM.

Certain embodiments comprise administering the at least one anti-NRP2antibody in an amount and at a frequency sufficient to achieve areduction in the circulating levels of HRS:NRP2 complexes, optionally areduction of about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 95, 99, or 100%.

In some embodiments, the at least one anti-NRP2 antibody enhances theimmune response to the cancer by about, or at least about, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000% or more, relative to a control.

In some embodiments, the at least one anti-NRP2 antibody reduces therate of in vitro growth of the cancer by about or at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000% or more relative to an untreatedcontrol.

In some embodiments, the at least one anti-NRP2 antibody reduces the invitro adhesiveness of the cancer to a substrate by about or at leastabout, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to anuntreated control. In some instances, the substrate comprises laminin.

In some embodiments, the at least one anti-NRP2 antibody reduces theinvasiveness of the cancer by about or at least about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000% or more relative to an untreated control.

In some embodiments, the at least one anti-NRP2 antibody inhibits therate of migration or motility of the cancer by about or at least about5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to anuntreated control.

In some embodiments, the at least one anti-NRP2 antibody inhibits therate of autophagy or endosome maturation (for example, endosomeacidification) of the cancer or associated immune cells by about or atleast about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative toan untreated control.

In some embodiments, the at least one anti-NRP2 antibody enhances thesusceptibility of the cancer to a chemotherapeutic agent, hormonaltherapeutic agent, or kinase inhibitor, by about or at least about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000% or more relative to thechemotherapeutic agent alone.

In some embodiments, the at least one anti-NRP2 antibody enhances ananti-tumor and/or immunostimulatory activity of the cancer immunotherapyagent by about, or at least about, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000% or more, relative to the cancer immunotherapy agent alone.

Certain embodiments comprise administering the at least one anti-NRP2antibody in an amount and at a frequency sufficient to achieve a steadystate concentration, or average circulating concentration, of the atleast one anti-NRP2 antibody of between about 1 nM and about 1 μM,between about 1 nM and about 100 nM, between about 1 nM and about 10 nM,or between about 1 nM and about 3 μM.

Also included are patient care kits, comprising:

(a) at least one antibody or antigen-binding fragment thereof thatspecifically binds to a human neuropilin-2 (NRP2) polypeptide, asdescribed herein; and optionally

(b) at least one additional agent selected from one or more of a cancerimmunotherapy agent, a chemotherapeutic agent, a hormonal therapeuticagent, and a kinase inhibitor.

In some embodiments, (a) and (b) are in separate therapeuticcompositions. In some embodiments, (a) and (b) are in the sametherapeutic composition, as described herein.

In some patient care kits, the at least one chemotherapeutic agent isselected from one or more of an alkylating agent, an anti-metabolite, acytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), andan anti-microtubule agent.

In some patient care kits, the alkylating agent is selected from one ormore of nitrogen mustards (optionally mechlorethamine, cyclophosphamide,mustine, melphalan, chlorambucil, ifosfamide, and busulfan),nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine(BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, andstreptozotocin), tetrazines (optionally dacarbazine, mitozolomide, andtemozolomide), aziridines (optionally thiotepa, mytomycin, anddiaziquone (AZQ)), cisplatins and derivatives thereof (optionallycarboplatin and oxaliplatin), and non-classical alkylating agents(optionally procarbazine and hexamethylmelamine);

the anti-metabolite is selected from one or more of anti-folates(optionally methotrexate and pemetrexed), fluoropyrimidines (optionally5-fluorouracil and capecitabine), deoxynucleoside analogues (optionallyancitabine, enocitabine, cytarabine, gemcitabine, decitabine,azacitidine, fludarabine, nelarabine, cladribine, clofarabine,fludarabine, and pentostatin), and thiopurines (optionally thioguanineand mercaptopurine);

the cytotoxic antibiotic is selected from one or more of anthracyclines(optionally doxorubicin, daunorubicin, epirubicin, idarubicin,pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C,mitoxantrone, and actinomycin;

the topoisomerase inhibitor is selected from one or more ofcamptothecin, irinotecan, topotecan, etoposide, doxorubicin,mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; and/or

the anti-microtubule agent is selected from one or more of taxanes(optionally paclitaxel and docetaxel) and vinca alkaloids (optionallyvinblastine, vincristine, vindesine, vinorelbine).

In some patient care kits, the at least one hormonal therapeutic agentis a hormonal agonist or a hormonal antagonist. In some patient carekits, the hormonal agonist is selected from one or more of a progestogen(progestin), a corticosteroid (optionally prednisolone,methylprednisolone, or dexamethasone), insulin like growth factors, VEGFderived angiogenic and lymphangiogenic factors (optionally VEGF-A,VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor(FGF), galectin, hepatocyte growth factor (HGF), platelet derived growthfactor (PDGF), transforming growth factor (TGF)-beta, an androgen, anestrogen, and a somatostatin analog.

In some patient care kits, the hormonal antagonist is selected from oneor more of a hormone synthesis inhibitor, optionally an aromataseinhibitor or a gonadotropin-releasing hormone (GnRH) or an analogthereof, and a hormone receptor antagonist, optionally a selectiveestrogen receptor modulator (SERM) or an anti-androgen, or an antibodydirected against a hormonal receptor, optionally cixutumumab,dalotuzumab, figitumumab, ganitumab, istiratumab, robatumumab,alacizumab pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab,metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab,imgatuzumab, laprituximab emtansine, matuzumab, modotuximab,necitumumab, nimotuzumab, panitumumab, tomuzotuximab, zalutumumab,aprutumab ixadotin, bemarituzumab, olaratumab, or tovetumab.

In some patient care kits, the kinase inhibitor is selected from one ormore of adavosertib, afanitib, aflibercept, axitinib, bevacizumab,bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib,entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib,imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab,pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib,sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, andvemuafenib

Also included are bioassay systems, comprising a substantially pureanti-NRP2 antibody or antigen-binding fragment thereof, optionally asdefined herein, and a host cell line that expresses a human NRP2polypeptide on the cell surface.

In some embodiments, the NRP2 polypeptide is labeled with a detectablelabel. In some embodiments, the anti-NRP2 antibody is labeled with adetectable label. In some embodiments, the NRP2 polypeptide isfunctionally coupled to a readout or indicator, such as a fluorescent orluminescent indicator of biological activity of the NRP2 polypeptide. Insome embodiments, the NRP2 polypeptide is selected from Table N1. Somebioassay systems comprise at least one NRP2 ligand (for example, an NRP2ligand from Table N2 or Table N3 and/or a human histidyl-tRNA synthetase(HRS) polypeptide from Table H1), for example, wherein the host cellexpresses the at least one NRP2 ligand. In some embodiments, the HRSpolypeptide is selected from Table H1, for example, wherein the HRSpolypeptide comprises a HRS splice variant, optionally selected fromHisRS^(N1), HisRS^(N2), HisRS^(N3), HisRS^(N4), HisRS^(N5), HisRS^(C1),HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5), HisRS^(C6), HisRS^(C7),HisRS^(C8), and HisRS^(C9). In some embodiments, the at least one NRP2ligand is selected from Table N2 or Table N3.

Some embodiments include a detection system, comprising a cell thatexpresses a human neuropilin 2 (NRP2) polypeptide and at least one NRP2ligand (for example, a recombinant NRP2 ligand selected from Table N2 orTable N3 and/or a human histidyl-tRNA synthetase (HRS) polypeptide fromTable H1), and a human or humanized anti-NRP2 antibody orantigen-binding fragment thereof, as described herein, which modulatesthe interaction between the NRP2 polypeptide and the at least one NRP2ligand. In some embodiments, the anti-NRP2 antibody is labeled with adetectable label. In some embodiments, the NRP2 polypeptide is selectedfrom Table N1. In some embodiments, the HRS polypeptide is selected fromTable H1, for example, wherein the HRS polypeptide comprises a HRSsplice variant, optionally selected from HisRS^(N1), HisRS^(N2),HisRS^(N3), HisRS^(N4), HisRS^(N5), HisRS^(C1), HisRS^(C2), HisRS^(C3),HisRS^(C4), HisRS^(C5), HisRS^(C6), HisRS^(C7), HisRS^(C8), andHisRS^(C9). In some embodiments, the at least one NRP2 ligand isselected from Table N2 or Table N3. In some embodiments, the NRP2polypeptide and/or the at least one NRP2 ligand (for example, an NRP2ligand from Table N2 or Table N3 and/or an HRS polypeptide from TableH1) is/are functionally coupled to a readout or indicator, such as afluorescent or luminescent indicator of biological activity of the NRP2polypeptide or the at least one NRP2 ligand.

Also included are diagnostic systems, comprising a cell that comprises aneuropilin 2 (NRP2) polypeptide, and at least one NRP2 ligand thatspecifically binds to the NRP2 polypeptide (for example, an NRP2 ligandselected from Table N2 or Table N3 and/or a human histidyl-tRNAsynthetase (HRS) polypeptide selected from Table H1), wherein the cellcomprises an indicator molecule that indicates a change in the levels oractivity of the NRP2 polypeptide in response to interaction with the atleast one NRP2 ligand.

Also included are cellular compositions, comprising an engineeredpopulation of cells in which at least one cell comprises one or morepolynucleotides encoding a human or humanized anti-NRP2 antibody orantigen-binding fragment thereof, as described herein, wherein the cellsare capable of growing in a serum-free medium.

Also included are cellular growth devices, comprising a human orhumanized anti-NRP2 antibody or antigen-binding fragment thereof, asdescribed herein, an engineered population of cells in which at leastone cell comprises one or more polynucleotides encoding said anti-NRP2antibody or antigen-binding fragment thereof, at least about 10 litersof a serum-free growth medium, and a sterile container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate the general domain structure of neuropilins (1A)and exemplary neuropilin co-receptor functions (1B).

FIG. 2 illustrates the domain structure of certain NRP2 isoforms andexemplary NRP2 ligand binding domains.

FIG. 3 shows a western blot of the indicated cell lines blotted with acommercially available antibody against human NRP2 (BAF2215, Boster,Calif.), using 6 μg of total cell lysate per lane. Molecular weightmakers are shown in the outer lanes.

FIGS. 4A-4D show flow cytometry cell surface binding plots on theindicated cell lines using the c domain specific anti-NRP2 antibody 3F2(aNRP2-2), which was detected using an AF647-conjugated goat anti-mouseIgG secondary antibody (GaM AF647). Binding of the secondary antibodyalone is shown in lighter grey (left curve in each Figure). FIG. 4Ashows antibody binding to U251 cells, FIG. 4B shows antibody binding toHUVEC cells, FIG. 4C shows antibody binding to THP-1 M1 cells, and FIG.4D shows antibody binding to HLEC cells.

FIGS. 5A-5D show flow cytometry surface binding plots to various celllines using the specified anti NRP2 antibodies. Antibody binding wasdetected using an AF647-conjugated goat anti-mouse IgG secondaryantibody (GaM AF647). FIG. 5A shows antibody binding to HUVEC cells withantibody concentrations ranging from 0.06 nM-1000 nM. FIG. 5B showsbinding to U251 cells, with antibody concentrations ranging from 0.01nM-1000 nM. FIG. 5C shows binding to A549 cells with antibodyconcentrations ranging from 0.01 nM-1000 nM. FIG. 5D shows binding toTHP-1 M1 cells, with antibody concentrations ranging from 0.01 nM-1000nM. Antibodies 14v2 (aNRP2-14), 10v2(aNRP2-10), 11v2(aNRP2-11),2v2(aNRP2-2), and isotype control (cMOPC21) were incubated with cells atthe indicated concentrations as described in the examples.

FIGS. 6A-6B show anti-NRP2 antibody binding curves to Expi293-hNRP2clonal cells over-expressing human NRP2. FIG. 6A shows antibodyconcentrations over the range 0.05-30 nM, and FIG. 6B shows the resultswith antibody concentrations tested over the range 0.02-10 nM, both witha 3-fold antibody dilution. Antibody binding was detected using anAF647-conjugated goat anti-mouse IgG secondary antibody (GaM AF647).Antibodies 8v2(aNRP2-8)#1328, 9v2(aNRP2-9)#1329, 10v2(aNRP2-10)#1330,14v2, 11v2(aNRP2-11)#1331, 14v2(aNRP2-14),#1344, 15v2(aNRP2-15)#1347 andisotype control (cMOPC21) (data not shown) were incubated with cells atthe indicated concentrations as described in the Examples.

FIGS. 7A-7B show the binding of VEGF-C to Expi293-hNRP2 clonal cellsover-expressing human NRP2. FIG. 7A shows the FACS binding curve usingVEGF-C(R&D systems), using rabbit detection antibody (Abcam) and a goatantibody AF647 labeled secondary antibody. FIG. 7B shows the flowcytometry scatter plots. From left to right, the curves in FIG. 7B show(a-R AF647); (Rb Iso_Ctl+a-R AF647); (a-VEGFc+a-R AF647); and (VEGFc 100nM+a-VEGFc+a-R AF647).

FIGS. 8A-8M show anti-NRP2 antibody blocking, displacement curves forthe indicated antibodies on VEGF-C binding to Expi293-hNRP2 clonal cellsover expressing human NRP2. FIG. 8A shows the results with the isotypecontrol antibody, FIG. 8B shows the results with antibody8v2(aNRP2-8)#1328, FIG. 8C shows the results with antibody9v2(aNRP2-9)#1329, FIG. 8D shows the results with antibody11v2(aNRP2-11)#1331, FIG. 8E shows the results with antibody14v2(aNRP2-14)#1344, FIG. 8F shows the results with antibody2v1(aNRP2-1)#1326, FIG. 8G shows the results with antibody2v2(aNRP2-2)#1327, FIG. 8H shows the results with antibody2v10(aNRP2-10)#1330, FIG. 8I shows the results with antibody #1333, FIG.8J shows the results with antibody #1334, FIG. 8K shows the results withantibody 2v7(aNRP2-7)#1335, FIG. 8L shows the results with antibody2v12(aNRP2-12)#1336, and FIG. 8M shows the results with antibody #1337.

FIG. 9 shows the binding of Sema3F-p95 and Sema 3F-p65 (0.05-100 nM) toExpi293-hNRP2 clonal cells over-expressing human NRP2. FIG. 9A shows theFACS binding curve using Sema 3F-p95 or p65 (in house), using ananti-myc detection antibody as described in the Examples.

FIGS. 10A-10H show anti-NRP2 antibody blocking, displacement curves forthe indicated antibodies on Sema 3F-p95 binding to Expi293-hNRP2 clonalcells over-expressing human NRP2. FIG. 10A shows the results with theisotype control antibody, FIG. 1013 shows the results with antibody14v2(aNRP2-14)#1344, FIG. 10C shows the results with antibody8v2(aNRP2-8)#1328, FIG. 10D shows the results with antibody9v2(aNRP2-9)#1329, FIG. 10E shows the results with antibody2v10(aNRP2-10)#1330, FIG. 10F shows the results with antibody15v2(aNRP2-15)#1347, FIG. 10G shows the results with antibody11v2(aNRP2-11)#1331, and FIG. 10H shows the results with antibody2v2(aNRP2-2)#1327.

FIGS. 11A-11F show anti-NRP2 antibody blocking, displacement curves forthe indicated antibodies on Sema 3F-p95 binding to Expi293-hNRP2 clonalcells over-expressing human NRP2. FIG. 11A shows the results withantibody 14v2(aNRP2-14)#1344, FIG. 11B shows the results with antibody8v2(aNRP2-8)#1328, FIG. 11C shows the results with antibody9v2(aNRP2-9)#1329, FIG. 11D shows the results with antibody2v10(aNRP2-10)#1330, FIG. 11E shows the results with antibody15v2(aNRP2-15)#1347, and FIG. 11F shows the results with antibody11v2(aNRP2-11)#1331.

FIG. 12 shows the effects of anti-NRP2 antibody binding on NRP2-plexinA1 induced receptor hetero-dimerization as described in the examples,using the disclosed antibodies.

FIG. 13 shows the effects of anti-NRP2 antibody binding on NRP2-FLT4(VEGFR3) induced receptor hetero dimerization as described in theexamples, using the disclosed antibodies.

FIG. 14 shows the crystal structure of human NRP2, showing the a2 domainand b1/b2 domains in complex with variable heavy (V_(H)) and light(V_(L)) chains of the aNRP2-14 Fab.

FIG. 15 shows the interaction surface between human NRP2 showing the a2domain and b1 domains and heavy (V_(H)) and light (V_(L)) chains of theaNRP2-14 Fab, and interacting amino acids.

FIG. 16 shows a view of the interaction interface between human NRP2 a2domain (lower) and the heavy (upper left) & light (upper right) chainsof the aNRP2-14 Fab. Interacting residues E237 of human NRP2 and S57 ofthe Fab heavy chain are circled.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any methods,materials, compositions, reagents, cells, similar or equivalent similaror equivalent to those described herein can be used in the practice ortesting of the subject matter of the present disclosure, preferredmethods and materials are described. All publications and references,including but not limited to patents and patent applications, cited inthis specification are herein incorporated by reference in theirentirety as if each individual publication or reference werespecifically and individually indicated to be incorporated by referenceherein as being fully set forth. Any patent application to which thisapplication claims priority is also incorporated by reference herein inits entirety in the manner described above for publications andreferences.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well-known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

For the purposes of the present disclosure, the following terms aredefined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” includes “one element”, “one ormore elements” and/or “at least one element”.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes. As used herein, the term “antigen”includes substances that are capable, under appropriate conditions, ofinducing an immune response to the substance and of reacting with theproducts of the immune response. For example, an antigen can berecognized by antibodies (humoral immune response) or sensitizedT-lymphocytes (T helper or cell-mediated immune response), or both.Antigens can be soluble substances, such as toxins and foreign proteins,or particulates, such as bacteria and tissue cells; however, only theportion of the protein or polysaccharide molecule known as the antigenicdeterminant (epitopes) combines with the antibody or a specific receptoron a lymphocyte. More broadly, the term “antigen” includes any substanceto which an antibody binds, or for which antibodies are desired,regardless of whether the substance is immunogenic. For such antigens,antibodies can be identified by recombinant methods, independently ofany immune response.

An “antagonist” refers to biological structure or chemical agent thatinterferes with or otherwise reduces the physiological action of anotheragent or molecule. In some instances, the antagonist specifically bindsto the other agent or molecule. Included are full and partialantagonists.

An “agonist” refers to biological structure or chemical agent thatincreases or enhances the physiological action of another agent ormolecule. In some instances, the agonist specifically binds to the otheragent or molecule. Included are full and partial agonists.

The term “anergy” refers to the functional inactivation of a T-cell, orB-cell response to re-stimulation by antigen.

As used herein, the term “amino acid” is intended to mean both naturallyoccurring and non-naturally occurring amino acids as well as amino acidanalogs and mimetics. Naturally-occurring amino acids include the 20(L)-amino acids utilized during protein biosynthesis as well as otherssuch as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,homocysteine, citrulline and ornithine, for example. Non-naturallyoccurring amino acids include, for example, (D)-amino acids, norleucine,norvaline, p-fluorophenylalanine, ethionine and the like, which areknown to a person skilled in the art. Amino acid analogs includemodified forms of naturally and non-naturally occurring amino acids.Such modifications can include, for example, substitution or replacementof chemical groups and moieties on the amino acid or by derivatizationof the amino acid. Amino acid mimetics include, for example, organicstructures which exhibit functionally similar properties such as chargeand charge spacing characteristic of the reference amino acid. Forexample, an organic structure which mimics arginine (Arg or R) wouldhave a positive charge moiety located in similar molecular space andhaving the same degree of mobility as the e-amino group of the sidechain of the naturally occurring Arg amino acid. Mimetics also includeconstrained structures so as to maintain optimal spacing and chargeinteractions of the amino acid or of the amino acid functional groups.Those skilled in the art know or can determine what structuresconstitute functionally equivalent amino acid analogs and amino acidmimetics.

As used herein, the term “antibody” encompasses not only intactpolyclonal or monoclonal antibodies, but also fragments thereof (such asdAb, Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), synthetic variantsthereof, naturally occurring variants, fusion proteins comprising anantibody portion with an antigen-binding fragment of the requiredspecificity, humanized antibodies, chimeric antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen-binding site or fragment (epitope recognition site) of therequired specificity. Certain features and characteristics of antibodies(and antigen-binding fragments thereof) are described in greater detailherein.

An antibody or antigen-binding fragment can be of essentially any type.As is well known in the art, an antibody is an immunoglobulin moleculecapable of specific binding to a target, such as an immune checkpointmolecule, through at least one epitope recognition site, located in thevariable region of the immunoglobulin molecule.

The term “antigen-binding fragment” as used herein refers to apolypeptide fragment that contains at least one CDR of an immunoglobulinheavy and/or light chain that binds to the antigen of interest. In thisregard, an antigen-binding fragment of the herein described antibodiesmay comprise 1, 2, 3, 4, 5, or all 6 CDRs of a V_(H) and V_(L) sequencefrom antibodies that bind to a target molecule.

The binding properties of antibodies and antigen-binding fragmentsthereof can be quantified using methods well known in the art (seeDavies et al., Annual Rev. Biochem. 59:439-473, 1990). In someembodiments, an antibody or antigen-binding fragment thereofspecifically binds to a target molecule, for example, an NRP2polypeptide or an epitope or complex thereof, with an equilibriumdissociation constant that is about or ranges from about ≤10⁻⁷ M toabout 10⁻⁸ M. In some embodiments, the equilibrium dissociation constantis about or ranges from about ≤10⁻⁹ M to about ≤10⁻¹⁰ M. In certainillustrative embodiments, an antibody or antigen-binding fragmentthereof has an affinity (Kd or EC₅₀) for a target molecule (to which itspecifically binds) of about, at least about, or less than about, 0.01,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, or 50 nM.

A molecule such as a polypeptide or antibody is said to exhibit“specific binding” or “preferential binding” if it reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell, substance, or particular epitope thanit does with alternative cells or substances, or epitopes. An antibody“specifically binds” or “preferentially binds” to a target molecule orepitope if it binds with greater affinity, avidity, more readily, and/orwith greater duration than it binds to other substances or epitopes, forexample, by a statistically significant amount. Typically one member ofthe pair of molecules that exhibit specific binding has an area on itssurface, or a cavity, which specifically binds to and is thereforecomplementary to a particular spatial and/or polar organization of theother member of the pair of molecules. Thus, the members of the pairhave the property of binding specifically to each other. For instance,an antibody that specifically or preferentially binds to a specificepitope is an antibody that binds that specific epitope with greateraffinity, avidity, more readily, and/or with greater duration than itbinds to other epitopes. It is also understood by reading thisdefinition that, for example, an antibody (or moiety or epitope) thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. The term is alsoapplicable where, for example, an antibody is specific for a particularepitope which is carried by a number of antigens, in which case thespecific binding member carrying the antigen-binding fragment or domainwill be able to bind to the various antigens carrying the epitope; forexample, it may be cross reactive to a number of different forms of atarget antigen from multiple species that share a common epitope

Immunological binding generally refers to the non-covalent interactionsof the type which occur between an immunoglobulin molecule and anantigen for which the immunoglobulin is specific, for example by way ofillustration and not limitation, as a result of electrostatic, ionic,hydrophilic and/or hydrophobic attractions or repulsion, steric forces,hydrogen bonding, van der Waals forces, and other interactions. Thestrength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (Kd) of the interaction,wherein a smaller Kd represents a greater affinity. Immunologicalbinding properties of selected polypeptides can be quantified usingmethods well known in the art. One such method entails measuring therates of antigen-binding site/antigen complex formation anddissociation, wherein those rates depend on the concentrations of thecomplex partners, the affinity of the interaction, and on geometricparameters that equally influence the rate in both directions. Thus,both the “on rate constant” (Kon) and the “off rate constant” (Koff) canbe determined by calculation of the concentrations and the actual ratesof association and dissociation. The ratio of Koff/Kon enablescancellation of all parameters not related to affinity, and is thusequal to the dissociation constant Kd. As used herein, the term“affinity” includes the equilibrium constant for the reversible bindingof two agents and is expressed as Kd or EC₅₀. Affinity of a bindingprotein to a ligand such as affinity of an antibody for an epitope canbe, for example, from about 100 nanomolar (nM) to about 0.1 nM, fromabout 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1femtomolar (fM). As used herein, the term “avidity” refers to theresistance of a complex of two or more agents to dissociation afterdilution. In some embodiments, affinity is expressed in the terms of thehalf maximal effective concentration (EC₅₀), which refers to theconcentration of an agent, such as an antibody, or an anti-NRP2antibody, as disclosed herein, which induces a response halfway betweenthe baseline and maximum after a specified exposure time. The EC₅₀ iscommonly used as a measure of an antibody's potency.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.Monoclonal antibodies specific for a polypeptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Also included aremethods that utilize transgenic animals such as mice to express humanantibodies. See, e.g., Neuberger et al., Nature Biotechnology 14:826,1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101,1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995.Particular examples include the VELOCIMMUNE® platform by REGENEREX®(see, e.g., U.S. Pat. No. 6,596,541).

Antibodies can also be generated or identified by the use of phagedisplay or yeast display libraries (see, e.g., U.S. Pat. No. 7,244,592;Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examplesof available libraries include cloned or synthetic libraries, such asthe Human Combinatorial Antibody Library (HuCAL), in which thestructural diversity of the human antibody repertoire is represented byseven heavy chain and seven light chain variable region genes. Thecombination of these genes gives rise to 49 frameworks in the masterlibrary. By superimposing highly variable genetic cassettes(CDRs=complementarity determining regions) on these frameworks, the vasthuman antibody repertoire can be reproduced. Also included are humanlibraries designed with human-donor-sourced fragments encoding alight-chain variable region, a heavy-chain CDR-3, synthetic DNA encodingdiversity in heavy-chain CDR-1, and synthetic DNA encoding diversity inheavy-chain CDR-2. Other libraries suitable for use will be apparent topersons skilled in the art.

In certain embodiments, antibodies and antigen-binding fragments thereofas described herein include a heavy chain and a light chain CDR set,respectively interposed between a heavy chain and a light chainframework region (FR) set which provide support to the CDRs and definethe spatial relationship of the CDRs relative to each other. As usedherein, the term “CDR set” refers to the three hypervariable regions ofa heavy or light chain V region. Proceeding from the N-terminus of aheavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and“CDR3” respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 orCDR3) is referred to herein as a “molecular recognition unit.”Crystallographic analysis of a number of antigen-antibody complexes hasdemonstrated that the amino acid residues of CDRs form extensive contactwith bound antigen, wherein the most extensive antigen contact is withthe heavy chain CDR3. Thus, the molecular recognition units areprimarily responsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

The structures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest. 4th Edition. US Department of Health and HumanServices. 1987, and updates thereof.

Also include are “monoclonal” antibodies, which refer to a homogeneousantibody population wherein the monoclonal antibody is comprised ofamino acids (naturally occurring and non-naturally occurring) that areinvolved in the selective binding of an epitope. Monoclonal antibodiesare highly specific, being directed against a single epitope. The term“monoclonal antibody” encompasses not only intact monoclonal antibodiesand full-length monoclonal antibodies, but also fragments thereof (suchas Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), variants thereof,fusion proteins comprising an antigen-binding portion, humanizedmonoclonal antibodies, chimeric monoclonal antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen-binding fragment (epitope recognition site) of the requiredspecificity and the ability to bind to an epitope. It is not intended tobe limited as regards the source of the antibody or the manner in whichit is made (e.g., by hybridoma, phage selection, recombinant expression,transgenic animals). The term includes whole immunoglobulins as well asthe fragments etc. described above under the definition of “antibody.”

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)2 fragment which comprises bothantigen-binding sites. An Fv fragment for use according to certainembodiments can be produced by preferential proteolytic cleavage of anIgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fvfragments are, however, more commonly derived using recombinanttechniques known in the art. The Fv fragment includes a non-covalentVH::VL heterodimer including an antigen-binding site which retains muchof the antigen recognition and binding capabilities of the nativeantibody molecule. See Inbar et al., PNAS USA. 69:2659-2662, 1972;Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al.,Biochem. 19:4091-4096, 1980.

In certain embodiments, single chain Fv (scFV) antibodies arecontemplated. For example, Kappa bodies (III et al., Prot. Eng.10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9, 1994);diabodies (Holliger et al., PNAS 90: 6444-8, 1993); or Janusins(Traunecker et al., EMBO J 10: 3655-59, 1991; and Traunecker et al.,Int. J. Cancer Suppl. 7:51-52, 1992), may be prepared using standardmolecular biology techniques following the teachings of the presentapplication with regard to selecting antibodies having the desiredspecificity.

A single chain Fv (scFv) polypeptide is a covalently linked VH::VLheterodimer which is expressed from a gene fusion including VH- andVL-encoding genes linked by a peptide-encoding linker. Huston et al.(PNAS USA. 85(16):5879-5883, 1988). A number of methods have beendescribed to discern chemical structures for converting the naturallyaggregated—but chemically separated—light and heavy polypeptide chainsfrom an antibody V region into an scFv molecule which will fold into athree dimensional structure substantially similar to the structure of anantigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405,to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.

In certain embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of a “diabody.” Diabodies are multimersof polypeptides, each polypeptide comprising a first domain comprising abinding region of an immunoglobulin light chain and a second domaincomprising a binding region of an immunoglobulin heavy chain, the twodomains being linked (e.g., by a peptide linker) but unable to associatewith each other to form an antigen-binding site: antigen-binding sitesare formed by the association of the first domain of one polypeptidewithin the multimer with the second domain of another polypeptide withinthe multimer (WO94/13804). A dAb fragment of an antibody consists of aVH domain (Ward et al., Nature 341:544-546, 1989). Diabodies and othermultivalent or multispecific fragments can be constructed, for example,by gene fusion (see WO94/13804; and Holliger et al., PNAS USA.90:6444-6448, 1993)).

Minibodies comprising a scFv joined to a CH3 domain are also included(see Hu et al., Cancer Res. 56:3055-3061, 1996). See also Ward et al.,Nature. 341:544-546, 1989; Bird et al., Science. 242:423-426, 1988;Huston et al., PNAS USA. 85:5879-5883, 1988); PCT/US92/09965;WO94/13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993),e.g., prepared chemically or from hybrid hybridomas, or may be any ofthe bispecific antibody fragments mentioned above. Diabodies and scFvcan be constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against antigen X, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(Ridgeway et al., Protein Eng., 9:616-621, 1996).

In certain embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of a UniBody®. A UniBody® is an IgG4antibody with the hinge region removed (see GenMab Utrecht, TheNetherlands; see also, e.g., US20090226421). This antibody technologycreates a stable, smaller antibody format with an anticipated longertherapeutic window than current small antibody formats. IgG4 antibodiesare considered inert and thus do not interact with the immune system.Fully human IgG4 antibodies may be modified by eliminating the hingeregion of the antibody to obtain half-molecule fragments having distinctstability properties relative to the corresponding intact IgG4 (GenMab,Utrecht). Halving the IgG4 molecule leaves only one area on the UniBody®that can bind to cognate antigens (e.g., disease targets) and theUniBody® therefore binds univalently to only one site on target cells.For certain cancer cell surface antigens, this univalent binding may notstimulate the cancer cells to grow as may be seen using bivalentantibodies having the same antigen specificity, and hence UniBody®technology may afford treatment options for some types of cancer thatmay be refractory to treatment with conventional antibodies. The smallsize of the UniBody® can be a great benefit when treating some forms ofcancer, allowing for better distribution of the molecule over largersolid tumors and potentially increasing efficacy.

In certain embodiments, the antibodies and antigen-binding fragmentsdescribed herein are in the form of a nanobody. Minibodies are encodedby single genes and are efficiently produced in almost all prokaryoticand eukaryotic hosts, for example, E. coli (see U.S. Pat. No.6,765,087), molds (for example Aspergillus or Trichoderma) and yeast(for example Saccharomyces, Kluyvermyces, Hansenula or Pichia (see U.S.Pat. No. 6,838,254). The production process is scalable andmulti-kilogram quantities of nanobodies have been produced. Nanobodiesmay be formulated as a ready-to-use solution having a long shelf life.The Nanoclone method (see WO 06/079372) is a proprietary method forgenerating Nanobodies against a desired target, based on automatedhigh-throughput selection of B-cells.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of an aptamer (see, e.g., Ellington etal., Nature. 346, 818-22, 1990; and Tuerk et al., Science. 249, 505-10,1990, incorporated by reference). Examples of aptamers included nucleicacid aptamers (e.g., DNA aptamers, RNA aptamers) and peptide aptamers.Nucleic acid aptamers refer generally to nucleic acid species that havebeen engineered through repeated rounds of in vitro selection orequivalent method, such as SELEX (systematic evolution of ligands byexponential enrichment), to bind to various molecular targets such assmall molecules, proteins, nucleic acids, and even cells, tissues andorganisms. See, e.g., U.S. Pat. Nos. 6,376,190; and 6,387,620,incorporated by reference.

Peptide aptamers typically include a variable peptide loop attached atboth ends to a protein scaffold, a double structural constraint thattypically increases the binding affinity of the peptide aptamer tolevels comparable to that of an antibody's (e.g., in the nanomolarrange). In certain embodiments, the variable loop length may be composedof about 10-20 amino acids (including all integers in between), and thescaffold may include any protein that has good solubility and compacityproperties. Certain exemplary embodiments utilize the bacterial proteinThioredoxin-A as a scaffold protein, the variable loop being insertedwithin the reducing active site (-Cys-Gly-Pro-Cys-loop in the wildprotein), with the two cysteines lateral chains being able to form adisulfide bridge. Methods for identifying peptide aptamers aredescribed, for example, in U.S. Application No. 2003/0108532,incorporated by reference. Peptide aptamer selection can be performedusing different systems known in the art, including the yeast two-hybridsystem.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of an avimer. Avimers refer tomultimeric binding proteins or peptides engineered using in vitro exonshuffling and phage display. Multiple binding domains are linked,resulting in greater affinity and specificity compared to single epitopeimmunoglobulin domains. See, e.g., Silverman et al., NatureBiotechnology. 23:1556-1561, 2005; U.S. Pat. No. 7,166,697; and U.S.Application Nos. 2004/0175756, 2005/0048512, 2005/0053973, 2005/0089932and 2005/0221384, incorporated by reference.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of an adnectin. Adnectins refer to aclass of targeted biologics derived from human fibronectin, an abundantextracellular protein that naturally binds to other proteins. See, e.g.,U.S. Application Nos. 2007/0082365; 2008/0139791; and 2008/0220049,incorporated by reference. Adnectins typically consists of a naturalfibronectin backbone, as well as the multiple targeting domains of aspecific portion of human fibronectin. The targeting domains can beengineered to enable an adnectin to specifically recognize an NRP2polypeptide or an epitope thereof.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of an anticalin. Anticalins refer to aclass of antibody mimetics that are typically synthesized from humanlipocalins, a family of binding proteins with a hypervariable loopregion supported by a structurally rigid framework. See, e.g., U.S.Application No. 2006/0058510. Anticalins typically have a size of about20 kDa. Anticalins can be characterized by a barrel structure formed byeight antiparallel β-strands (a stable β-barrel scaffold) that arepairwise connected by four peptide loops and an attached α-helix. Incertain aspects, conformational deviations to achieve specific bindingare made in the hypervariable loop region(s). See, e.g., Skerra, FEBS J.275:2677-83, 2008, incorporated by reference.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of a designed ankyrin repeat protein(DARPin). DARPins include a class of non-immunoglobulin proteins thatcan offer advantages over antibodies for target binding in drugdiscovery and drug development. Among other uses, DARPins are ideallysuited for in vivo imaging or delivery of toxins or other therapeuticpayloads because of their favorable molecular properties, includingsmall size and high stability. The low-cost production in bacteria andthe rapid generation of many target-specific DARPins make the DARPinapproach useful for drug discovery. Additionally, DARPins can be easilygenerated in multispecific formats, offering the potential to target aneffector DARPin to a specific organ or to target multiple receptors withone molecule composed of several DARPins. See, e.g., Stumpp et al., CurrOpin Drug Discov Devel. 10:153-159, 2007; U.S. Application No.2009/0082274; and PCT/EP2001/10454, incorporated by reference.

Also included are heavy chain dimers, such as antibodies from camelidsand sharks. Camelid and shark antibodies comprise a homodimeric pair oftwo chains of V-like and C-like domains (neither has a light chain).Since the VH region of a heavy chain dimer IgG in a camelid does nothave to make hydrophobic interactions with a light chain, the region inthe heavy chain that normally contacts a light chain is changed tohydrophilic amino acid residues in a camelid. VH domains of heavy-chaindimer IgGs are called VHH domains. Shark Ig-NARs comprise a homodimer ofone variable domain (termed a V-NAR domain) and five C-like constantdomains (C-NAR domains).

In camelids, the diversity of antibody repertoire is determined by thecomplementary determining regions (CDR) 1, 2, and 3 in the VH or VHHregions. The CDR3 in the camel VHH region is characterized by itsrelatively long length averaging 16 amino acids (Muyldermans et al.,1994, Protein Engineering 7(9): 1129). This is in contrast to CDR3regions of antibodies of many other species. For example, the CDR3 ofmouse VH has an average of 9 amino acids. Libraries of camelid-derivedantibody variable regions, which maintain the in vivo diversity of thevariable regions of a camelid, can be made by, for example, the methodsdisclosed in U.S. Patent Application Ser. No. 20050037421, publishedFeb. 17, 2005

In certain embodiments, the antibodies or antigen-binding fragmentsthereof are humanized. These embodiments refer to a chimeric molecule,generally prepared using recombinant techniques, having anantigen-binding site derived from an immunoglobulin from a non-humanspecies and the remaining immunoglobulin structure of the molecule basedupon the structure and/or sequence of a human immunoglobulin. Theantigen-binding site may comprise either complete variable domains fusedonto constant domains or only the CDRs grafted onto appropriateframework regions in the variable domains. Epitope binding sites may bewild type or modified by one or more amino acid substitutions. Thiseliminates the constant region as an immunogen in human individuals, butthe possibility of an immune response to the foreign variable regionremains (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al.,PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327,1988). Illustrative methods for humanization of antibodies include themethods described in U.S. Pat. No. 7,462,697.

Another approach focuses not only on providing human-derived constantregions, but modifying the variable regions as well so as to reshapethem as closely as possible to human form. It is known that the variableregions of both heavy and light chains contain threecomplementarity-determining regions (CDRs) which vary in response to theepitopes in question and determine binding capability, flanked by fourframework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennonhuman antibodies are prepared with respect to a particular epitope,the variable regions can be “reshaped” or “humanized” by grafting CDRsderived from nonhuman antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann etal., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536,1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maedaet al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNASUSA. 88:4181-4185, 1991; Tempest et al., Bio/Technology 9:266-271, 1991;Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA.89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. Insome embodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, six) which are altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody.

In certain embodiments, the antibodies are “chimeric” antibodies. Inthis regard, a chimeric antibody is comprised of an antigen-bindingfragment of an antibody operably linked or otherwise fused to aheterologous Fc portion of a different antibody. In certain embodiments,the Fc domain or heterologous Fc domain is of human origin. In certainembodiments, the Fc domain or heterologous Fc domain is of mouse origin.In other embodiments, the heterologous Fc domain may be from a differentIg class from the parent antibody, including IgA (including subclassesIgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3,and IgG4), and IgM. In further embodiments, the heterologous Fc domainmay be comprised of CH2 and CH3 domains from one or more of thedifferent Ig classes. As noted above with regard to humanizedantibodies, the antigen-binding fragment of a chimeric antibody maycomprise only one or more of the CDRs of the antibodies described herein(e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), ormay comprise an entire variable domain (VL, VH or both).

As used herein, a subject “at risk” of developing a disease, or adversereaction may or may not have detectable disease, or symptoms of disease,and may or may not have displayed detectable disease or symptoms ofdisease prior to the treatment methods described herein. “At risk”denotes that a subject has one or more risk factors, which aremeasurable parameters that correlate with development of a disease, asdescribed herein and known in the art. A subject having one or more ofthese risk factors has a higher probability of developing disease, or anadverse reaction than a subject without one or more of these riskfactor(s).

“Biocompatible” refers to materials or compounds which are generally notinjurious to biological functions of a cell or subject and which willnot result in any degree of unacceptable toxicity, including allergenicand disease states.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notdirectly contribute to the code for the polypeptide product of a gene.

Throughout this disclosure, unless the context requires otherwise, thewords “comprise,” “comprises,” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

The term “effector function”, or “ADCC effector function” in the contextof antibodies refers to the ability of that antibody to engage withother arms of the immune system, including for example, the activationof the classical complement pathway, or through engagement of Fcreceptors. Complement dependent pathways are primarily driven by theinteraction of C1q with the C1 complex with clustered antibody Fcdomains. Antibody dependent cellular cytotoxicity (ADCC), is primarilydriven by the interaction of Fc receptors (FcRs) on the surface ofeffector cells (natural killer cells, macrophages, monocytes andeosinophils) which bind to the Fc region of an IgG which itself is boundto a target cell. Fc receptors (FcRs) are key immune regulatoryreceptors connecting the antibody mediated (humoral) immune response tocellular effector functions. Receptors for all classes ofimmunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE),FcαRI (IgA), FcμR (IgM) and FcδR (IgD). There are at least three classesof receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32(FcγRIIa, FcγRIIb and FcγRIIc) and CD16 (FcγRIIIa and FcγRIIIb). FcγRIis classed as a high affinity receptor (nanomolar range KD) while FcγRIIand FcγRIII are low to intermediate affinity (micromolar range KD). UponFc binding a signaling pathway is triggered which results in thesecretion of various substances, such as lytic enzymes, perforin,granzymes and tumour necrosis factor, which mediate in the destructionof the target cell. The level of ADCC effector function various forhuman IgG subtypes. Although this is dependent on the allotype andspecific FcvR, in simple terms ADCC effector function is “high” forhuman IgG1 and IgG3, and “low” for IgG2 and IgG4.

The term “endotoxin free” or “substantially endotoxin free” relatesgenerally to compositions, solvents, and/or vessels that contain at mosttrace amounts (e.g., amounts having no clinically adverse physiologicaleffects to a subject) of endotoxin, and preferably undetectable amountsof endotoxin. Endotoxins are toxins associated with certainmicro-organisms, such as bacteria, typically gram-negative bacteria,although endotoxins may be found in gram-positive bacteria, such asListeria monocytogenes. The most prevalent endotoxins arelipopolysaccharides (LPS) or lipo-oligo-saccharides (LOS) found in theouter membrane of various Gram-negative bacteria, and which represent acentral pathogenic feature in the ability of these bacteria to causedisease. Small amounts of endotoxin in humans may produce fever, alowering of the blood pressure, and activation of inflammation andcoagulation, among other adverse physiological effects.

Therefore, in pharmaceutical production, it is often desirable to removemost or all traces of endotoxin from drug products and/or drugcontainers, because even small amounts may cause adverse effects inhumans. A depyrogenation oven may be used for this purpose, astemperatures in excess of 300° C. are typically required to break downmost endotoxins. For instance, based on primary packaging material suchas syringes or vials, the combination of a glass temperature of 250° C.and a holding time of 30 minutes is often sufficient to achieve a 3 logreduction in endotoxin levels. Other methods of removing endotoxins arecontemplated, including, for example, chromatography and filtrationmethods, as described herein and known in the art.

Endotoxins can be detected using routine techniques known in the art.For example, the Limulus Amoebocyte Lysate assay, which utilizes bloodfrom the horseshoe crab, is a very sensitive assay for detectingpresence of endotoxin. In this test, very low levels of LPS can causedetectable coagulation of the limulus lysate due a powerful enzymaticcascade that amplifies this reaction. Endotoxins can also be quantitatedby enzyme-linked immunosorbent assay (ELISA). To be substantiallyendotoxin free, endotoxin levels may be less than about 0.001, 0.005,0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2,2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mg of active compound. Typically, 1ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. An epitope includes a region of an antigen that is bound by anantibody. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl or sulfonyl, and may in certainembodiments have specific three-dimensional structural characteristics,and/or specific charge characteristics. Epitopes can be contiguous ornon-contiguous in relation to the primary structure of the antigen, forexample, an NRP2 polypeptide. In particular embodiments, an epitopecomprises, consists, or consists essentially of about, at least about,or no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 contiguous amino acids (i.e., a linear epitope) ornon-contiguous amino acids (i.e., conformational epitope) of a referencesequence (see, e.g., Table N1) or target molecule described herein.

An “epitope” includes that portion of an antigen or other macromoleculecapable of forming a binding interaction that interacts with thevariable region binding pocket of a binding protein. Such bindinginteraction can be manifested as an intermolecular contact with one ormore amino acid residues of a CDR. Antigen binding can involve a CDR3 ora CDR3 pair. An epitope can be a linear peptide sequence (i.e.,“continuous”) or can be composed of noncontiguous amino acid sequences(i.e., “conformational” or “discontinuous”). A binding protein canrecognize one or more amino acid sequences; therefore an epitope candefine more than one distinct amino acid sequence. Epitopes recognizedby binding protein can be determined by peptide mapping and sequenceanalysis techniques well known to one of skill in the art. A “crypticepitope” or a “cryptic binding site” is an epitope or binding site of aprotein sequence that is not exposed or substantially protected fromrecognition within an unmodified polypeptide, but is capable of beingrecognized by a binding protein of a denatured or proteolyzedpolypeptide. Amino acid sequences that are not exposed, or are onlypartially exposed, in the unmodified polypeptide structure are potentialcryptic epitopes. If an epitope is not exposed, or only partiallyexposed, then it is likely that it is buried within the interior of thepolypeptide. Candidate cryptic epitopes can be identified, for example,by examining the three-dimensional structure of an unmodifiedpolypeptide.

The term “half maximal effective concentration” or “EC₅₀” refers to theconcentration of an agent (e.g., antibody) as described herein at whichit induces a response halfway between the baseline and maximum aftersome specified exposure time; the EC₅₀ of a graded dose response curvetherefore represents the concentration of a compound at which 50% of itsmaximal effect is observed. EC50 also represents the plasmaconcentration required for obtaining 50% of a maximum effect in vivo.Similarly, the “EC₉₀” refers to the concentration of an agent orcomposition at which 90% of its maximal effect is observed. The “EC₉₀”can be calculated from the “EC50” and the Hill slope, or it can bedetermined from the data directly, using routine knowledge in the art.In some embodiments, the EC50 of an agent (e.g., antibody) is less thanabout 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 200 or 500 nM. In some embodiments, anagent will have an EC₅O value of about 1 nM or less.

“Immune response” means any immunological response originating fromimmune system, including responses from the cellular and humeral, innateand adaptive immune systems. Exemplary cellular immune cells include forexample, lymphocytes, macrophages, T cells, B cells, NK cells,neutrophils, eosinophils, dendritic cells, mast cells, monocytes, andall subsets thereof. Cellular responses include for example, effectorfunction, cytokine release, phagocytosis, efferocytosis, translocation,trafficking, proliferation, differentiation, activation, repression,cell-cell interactions, apoptosis, etc. Humeral responses include forexample IgG, IgM, IgA, IgE, responses and their corresponding effectorfunctions.

The “half-life” of an agent such as an antibody can refer to the time ittakes for the agent to lose half of its pharmacologic, physiologic, orother activity, relative to such activity at the time of administrationinto the serum or tissue of an organism, or relative to any otherdefined time-point. “Half-life” can also refer to the time it takes forthe amount or concentration of an agent to be reduced by half of astarting amount administered into the serum or tissue of an organism,relative to such amount or concentration at the time of administrationinto the serum or tissue of an organism, or relative to any otherdefined time-point. The half-life can be measured in serum and/or anyone or more selected tissues.

The terms “modulating” and “altering” include “increasing,” “enhancing”or “stimulating,” as well as “decreasing” or “reducing,” typically in astatistically significant or a physiologically significant amount ordegree relative to a control. An “increased,” “stimulated” or “enhanced”amount is typically a “statistically significant” amount, and mayinclude an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times (e.g., 500, 1000times) (including all integers and ranges in between e.g., 1.5, 1.6,1.7. 1.8, etc.) the amount produced by no composition (e.g., the absenceof agent) or a control composition. A “decreased” or “reduced” amount istypically a “statistically significant” amount, and may include a 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% decrease (including all integers and rangesin between) in the amount produced by no composition (e.g., the absenceof an agent) or a control composition. Examples of comparisons and“statistically significant” amounts are described herein.

The term “migratory cells” refers to cells that are capable of movementfrom one place to another in response to a stimulus. Exemplary migratorycells include immune cells such as monocytes, Natural Killer (NK) cells,dendritic cells (immature or mature), subsets of dendritic cellsincluding myeloid, plasmacytoid (also called lymphoid) and Langerhanscells, macrophages such as histiocytes, tissue resident macrophages suchas Kupffer's cells, microglia cells in the CNS, alveolar macrophages,and peritoneal macrophages, macrophage subtypes such as M0, M1, Mox,M2a, M2b, and M2c macrophages, neutrophils, eosinophils, mast cells,basophils, B cells including plasma B cells, memory B cells, B-1 cells,and B-2 cells, CD45RO (naive T) cells, CD45RA (memory T) cells, CD4Helper T Cells including Th1, Th2, and Tr1/Th3 cells, CD8 Cytotoxic TCells, Regulatory T Cells, Gamma Delta T Cells, and thymocytes.Additional examples of migratory cells include fibroblasts, fibrocytes,tumor cells, and stem cells. The term “cell migration” refers to themovement of migratory cells, and the term “modulation of cell migration”refers to the modulation of the movement of any such migratory cells.

The terms “polypeptide,” “protein” and “peptide” are usedinterchangeably and mean a polymer of amino acids not limited to anyparticular length. The term “enzyme” includes polypeptide or proteincatalysts. The terms include modifications such as myristoylation,sulfation, glycosylation, phosphorylation and addition or deletion ofsignal sequences. The terms “polypeptide” or “protein” means one or morechains of amino acids, wherein each chain comprises amino acidscovalently linked by peptide bonds, and wherein said polypeptide orprotein can comprise a plurality of chains non-covalently and/orcovalently linked together by peptide bonds, having the sequence ofnative proteins, that is, proteins produced by naturally-occurring andspecifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.In certain embodiments, the polypeptide is a “recombinant” polypeptide,produced by recombinant cell that comprises one or more recombinant DNAmolecules, which are typically made of heterologous polynucleotidesequences or combinations of polynucleotide sequences that would nototherwise be found in the cell.

The term “polynucleotide” and “nucleic acid” includes mRNA, RNA, cRNA,cDNA, and DNA. The term typically refers to polymeric form ofnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA. The terms“isolated DNA” and “isolated polynucleotide” and “isolated nucleic acid”refer to a molecule that has been isolated free of total genomic DNA ofa particular species. Therefore, an isolated DNA segment encoding apolypeptide refers to a DNA segment that contains one or more codingsequences yet is substantially isolated away from, or purified freefrom, total genomic DNA of the species from which the DNA segment isobtained. Also included are non-coding polynucleotides (e.g., primers,probes, oligonucleotides), which do not encode a polypeptide. Alsoincluded are recombinant vectors, including, for example, expressionvectors, viral vectors, plasmids, cosmids, phagemids, phage, viruses,and the like.

Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide described herein, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials. Hence,a polynucleotide or expressible polynucleotides, regardless of thelength of the coding sequence itself, may be combined with othersequences, for example, expression control sequences.

“Expression control sequences” include regulatory sequences of nucleicacids, or the corresponding amino acids, such as promoters, leaders,enhancers, introns, recognition motifs for RNA, or DNA binding proteins,polyadenylation signals, terminators, internal ribosome entry sites(IRES), secretion signals, subcellular localization signals, and thelike, which have the ability to affect the transcription or translation,or subcellular, or cellular location of a coding sequence in a hostcell. Exemplary expression control sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990).

A “promoter” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. As used herein, the promoter sequence isbounded at its 3′ terminus by the transcription initiation site andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to initiate transcription at levels detectableabove background. A transcription initiation site (conveniently definedby mapping with nuclease S1) can be found within a promoter sequence, aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. Eukaryotic promoters can often, but notalways, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoterscontain Shine-Dalgarno sequences in addition to the −10 and −35consensus sequences.

A large number of promoters, including constitutive, inducible andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources include for example, viral,mammalian, insect, plant, yeast, and bacterial cell types), and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available on line or, forexample, from depositories such as the ATCC as well as other commercialor individual sources. Promoters can be unidirectional (i.e., initiatetranscription in one direction) or bi-directional (i.e., initiatetranscription in either a 3′ or 5′ direction). Non-limiting examples ofpromoters include, for example, the T7 bacterial expression system, pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter,the SV40 promoter, the RSV promoter. Inducible promoters include the Tetsystem, (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone induciblesystem (No et al., Proc. Natl. Acad. Sci. (1996) 93 (8): 3346-3351; theT-REx™ system (Invitrogen Carlsbad, Calif.), LacSwitch® (Stratagene,(San Diego, Calif.) and the Cre-ERT tamoxifen inducible recombinasesystem (Indra et al. Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc.Acid. Res. (2000) 28 (23): e99; U.S. Pat. No. 7,112,715; and Kramer &Fussenegger, Methods Mol. Biol. (2005) 308: 123-144) or any promoterknown in the art suitable for expression in the desired cells.

An “expressible polynucleotide” includes a cDNA, RNA, mRNA or otherpolynucleotide that comprises at least one coding sequence andoptionally at least one expression control sequence, for example, atranscriptional and/or translational regulatory element, and which canexpress an encoded polypeptide upon introduction into a cell, forexample, a cell in a subject.

Various viral vectors that can be utilized to deliver an expressiblepolynucleotide include adenoviral vectors, herpes virus vectors,vaccinia virus vectors, adeno-associated virus (AAV) vectors, andretroviral vectors. In some instances, the retroviral vector is aderivative of a murine or avian retrovirus, or is a lentiviral vector.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number ofadditional retroviral vectors can incorporate multiple genes. All ofthese vectors can transfer or incorporate a gene for a selectable markerso that transduced cells can be identified and generated. By inserting apolypeptide sequence of interest into the viral vector, along withanother gene that encodes the ligand for a receptor on a specific targetcell, for example, the vector may be made target specific. Retroviralvectors can be made target specific by inserting, for example, apolynucleotide encoding a protein. Illustrative targeting may beaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector.

In particular embodiments, the expressible polynucleotide is a modifiedRNA or modified mRNA polynucleotide, for example, a non-naturallyoccurring RNA analog. In certain embodiments, the modified RNA or mRNApolypeptide comprises one or more modified or non-natural bases, forexample, a nucleotide base other than adenine (A), guanine (G), cytosine(C), thymine (T), and/or uracil (U). In some embodiments, the modifiedmRNA comprises one or more modified or non-natural internucleotidelinkages. Expressible RNA polynucleotides for delivering an encodedtherapeutic polypeptide are described, for example, in Kormann et al.,Nat Biotechnol. 29:154-7, 2011; and U.S. Application Nos. 2015/0111248;2014/0243399; 2014/0147454; and 2013/0245104, which are incorporated byreference in their entireties.

The term “isolated” polypeptide or protein referred to herein means thata subject protein (1) is free of at least some other proteins with whichit would typically be found in nature, (2) is essentially free of otherproteins from the same source, e.g., from the same species, (3) isexpressed by a cell from a different species, (4) has been separatedfrom at least about 50 percent of polynucleotides, lipids,carbohydrates, or other materials with which it is associated in nature,(5) is not associated (by covalent or non-covalent interaction) withportions of a protein with which the “isolated protein” is associated innature, (6) is operably associated (by covalent or non-covalentinteraction) with a polypeptide with which it is not associated innature, or (7) does not occur in nature. Such an isolated protein can beencoded by genomic DNA, cDNA, mRNA or other RNA, of may be of syntheticorigin, or any combination thereof. In certain embodiments, the isolatedprotein is substantially free from proteins or polypeptides or othercontaminants that are found in its natural environment that wouldinterfere with its use (therapeutic, diagnostic, prophylactic, researchor otherwise).

In certain embodiments, the “purity” of any given agent (e.g.,polypeptide such as an antibody) in a composition may be defined. Forinstance, certain compositions may comprise an agent such as apolypeptide agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% pure on a protein basis or aweight-weight basis, including all decimals and ranges in between, asmeasured, for example and by no means limiting, by high performanceliquid chromatography (HPLC), a well-known form of column chromatographyused frequently in biochemistry and analytical chemistry to separate,identify, and quantify compounds.

A “lipid nanoparticle” or “solid lipid nanoparticle” refers to one ormore spherical nanoparticles with an average diameter of between about10 to about 1000 nanometers, and which comprise a solid lipid corematrix that can solubilize lipophilic molecules. The lipid core isstabilized by surfactants (e.g., emulsifiers), and can comprise one ormore of triglycerides (e.g., tristearin), diglycerides (e.g., glycerolbahenate), monoglycerides (e.g., glycerol monostearate), fatty acids(e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g.,cetyl palmitate), including combinations thereof. Lipid nanoparticlesare described, for example, in Petrilli et al., Curr Pharm Biotechnol.15:847-55, 2014; and U.S. Pat. Nos. 6,217,912; 6,881,421; 7,402,573;7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750; 8,871,509;9,017,726; 9,173,853; 9,220,779; 9,227,917; and 9,278,130, which areincorporated by reference in their entireties. Certain compositionsdescribed herein are formulated with one or more lipid nanoparticles.

The terms or “Neuropilin 2-associated disease” or “NRP2-associateddisease” refer to diseases and conditions in which NRP2 activity,expression, and/or spatial distribution plays a role in thepathophysiology of that disease or condition. In some instances, NRP2associated diseases are modulated by the anti-NRP2 antibodies of thepresent disclosure by altering the interaction of NRP2 with at least oneNRP2 ligand to impact NRP2 activity, signaling, expression, and/orspatial distribution. Exemplary NRP2-associated diseases and conditionsinclude without limitation, cancer and diseases or pathologiesassociated with cancer including cancer cell growth, cancer initiation,cancer migration, cancer cell adhesion, invasion, and metastasis. Alsoincluded are diseases associated with inflammation and autoimmunity, andrelated inflammatory diseases, including disease associated withinappropriate immune cell activation or migration such as graft versushost disease (GVHD). Additional examples include diseases associatedwith lymphatic development, lymphangiogenesis, and lymphatic damage,including edema, lymphedema, secondary lymphedema, inappropriate fatabsorption and deposition, excess fat deposition, and vascularpermeability. Also included are diseases associated with infectionsincluding latent infections, and diseases associated with allergicdisorders/diseases and allergic responses, including chronic obstructivepulmonary disorder (COPD), neutrophilic asthma, antineutrophilcytoplasmic antibody (ANCA)-associated systemic vasculitis, systemiclupus erythematosus, rheumatoid arthritis, inflammasome-relateddisease(s), and skin-related neutrophil-mediated disease(s) such aspyoderma gangrenosum. Additional examples include diseases associatedwith granulomatous inflammatory diseases including sarcoidosis andgranulomas, and fibrotic diseases including endometriosis, fibrosis,endothelial to mesenchymal transition (EMT), and wound healing, amongothers. Also included are diseases associated with inappropriate smoothmuscle contractility and vascular smooth muscle cell migration and/oradhesion, and diseases associated with inappropriate autophagy,phagocytosis, and efferocytosis. Also included are diseases associatedwith inappropriate migratory cell movement, as described herein.Additional examples include neuronal diseases, including diseasesassociated with peripheral nervous system remodeling and painperception. Also included are diseases associated with bone developmentand/or bone remodeling. Typically, the term “inappropriate” refers to anactivity or characteristic that associates with or causes a pathology ordisease state.

The term “reference sequence” refers generally to a nucleic acid codingsequence, or amino acid sequence, to which another sequence is beingcompared. All polypeptide and polynucleotide sequences described hereinare included as references sequences, including those described by nameand those described in the Tables and the Sequence Listing.

Certain embodiments include biologically active “variants” and“fragments” of the polypeptides (e.g., antibodies) described herein, andthe polynucleotides that encode the same. “Variants” contain one or moresubstitutions, additions, deletions, and/or insertions relative to areference polypeptide or polynucleotide (see, e.g., the Tables and theSequence Listing). A variant polypeptide or polynucleotide comprises anamino acid or nucleotide sequence with at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity or similarity or homology to a referencesequence, as described herein, and substantially retains the activity ofthat reference sequence. Also included are sequences that consist of ordiffer from a reference sequences by the addition, deletion, insertion,or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140,150 or more amino acids or nucleotides and which substantially retainthe activity of that reference sequence. In certain embodiments, theadditions or deletions include C-terminal and/or N-terminal additionsand/or deletions.

The terms “sequence identity” or, for example, comprising a “sequence50% identical to,” as used herein, refer to the extent that sequencesare identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerized implementations of algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Drive Madison,Wis., USA) or by inspection and the best alignment (i.e., resulting inthe highest percentage homology over the comparison window) generated byany of the various methods selected. Reference also may be made to theBLAST family of programs as for example disclosed by Altschul et al.,Nucl. Acids Res. 25:3389, 1997.

The term “solubility” refers to the property of an agent (e.g.,antibody) provided herein to dissolve in a liquid solvent and form ahomogeneous solution. Solubility is typically expressed as aconcentration, either by mass of solute per unit volume of solvent (g ofsolute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity,molality, mole fraction or other similar descriptions of concentration.The maximum equilibrium amount of solute that can dissolve per amount ofsolvent is the solubility of that solute in that solvent under thespecified conditions, including temperature, pressure, pH, and thenature of the solvent. In certain embodiments, solubility is measured atphysiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0,pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certainembodiments, solubility is measured in water or a physiological buffersuch as PBS or NaCl (with or without NaPO₄). In specific embodiments,solubility is measured at relatively lower pH (e.g., pH 6.0) andrelatively higher salt (e.g., 500 mM NaCl and 10 mM NaPO₄). In certainembodiments, solubility is measured in a biological fluid (solvent) suchas blood or serum. In certain embodiments, the temperature can be aboutroom temperature (e.g., about 20, 21, 22, 23, 24, 25° C.) or about bodytemperature (37° C.). In certain embodiments, an agent has a solubilityof at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,40, 50, 60, 70, 80, 90 or 100 mg/ml at room temperature or at 37° C.

A “subject” or a “subject in need thereof” or a “patient” or a “patientin need thereof” includes a mammalian subject such as a human subject.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur, if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less.

“Therapeutic response” refers to improvement of symptoms (whether or notsustained) based on administration of one or more therapeutic agents.

As used herein, the terms “therapeutically effective amount”,“therapeutic dose,” “prophylactically effective amount,” or“diagnostically effective amount” is the amount of an agent (e.g.,anti-NRP2 antibody, immunotherapy agent) needed to elicit the desiredbiological response following administration.

As used herein, “treatment” of a subject (e.g., a mammal, such as ahuman) or a cell is any type of intervention used in an attempt to alterthe natural course of the individual or cell. Treatment includes, but isnot limited to, administration of a pharmaceutical composition, and maybe performed either prophylactically or subsequent to the initiation ofa pathologic event or contact with an etiologic agent. Also included are“prophylactic” treatments, which can be directed to reducing the rate ofprogression of the disease or condition being treated, delaying theonset of that disease or condition, or reducing the severity of itsonset. “Treatment” or “prophylaxis” does not necessarily indicatecomplete eradication, cure, or prevention of the disease or condition,or associated symptoms thereof.

The term “wild-type” refers to a gene or gene product (e.g., apolypeptide) that is most frequently observed in a population and isthus arbitrarily designed the “normal” or “wild-type” form of the gene.

Each embodiment in this specification is to be applied to every otherembodiment unless expressly stated otherwise.

Anti-NRP2 Antibodies

Certain embodiments include antibodies, and antigen-binding fragmentsthereof, which specifically bind to a human neuropilin 2 (NRP2)polypeptide. In some embodiments, the at least one antibody orantigen-binding fragment thereof modulates (e.g., interferes with)binding of the human NRP2 polypeptide to at least one NRP2 ligand, suchas a human histidyl-tRNA synthetase (HRS) polypeptide or other NRP2ligand.

Neuropilin-2 is a cell surface receptor protein that modulates a broadrange of cellular functions through its roles as an essential cellsurface receptor and co-receptor for a variety of ligands (see, e.g.,Guo and Vander Kooi, J. Cell. Biol. 290 No 49: 29120-29126. 2015). Forinstance, it functions during epithelial to mesenchymal transition(EMT), for example, by promoting TGF-β1-mediated EMT in colorectal andother cancer cells (see, e.g., Grandclement et al., PLoS ONE 6(7)e20444, 2011), and by mediating EMT or endo-EMT in fibroblasts,myofibroblasts, and endothelial cells to promote fibrosis formation(see, e.g., Pardali et al., Int. J. Mol. Sci. 18:2157, 2017).

Neuropilin-2 expression promotes lymphangiogenesis (see, e.g., Doci etal., Cancer Res. 75:2937-2948, 2015) single nucleotide polymorphisms(SNPs) in NRP2 are associated with lymphedema (see, e.g., Miaskowski etal., PLoS ONE 8(4) e60164, 2013). NRP2 also regulates smooth musclecontractility (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559,2012), regulates autophagy, for example, in cancer (see, e.g., Stantonet al., Cancer Res. 73:160-171, 2013), contributes to tumor initiation,survival, and metastasis (see, e.g., Goel et al., EMBO Mol. Med.5:488-508, 2013; and Samuel et al., PLoS ONE 6(10) e23208, 2011), andregulates immune cell activation and migration (see, e.g.,Mendes-da-Cruz et al., PLoS ONE 9(7) e103405, 2014). Neuropilins arealso multifunctional co-receptors involved in tumor initiation, growth,metastasis and immunity (see, e.g., Prud'homme et al., Oncotarget3:921-939, 2012).

Neuropilin-2 is expressed in various cells of the immune system,including lymphoid cells such as B and T cells, and myeloid cells suchas basophils, eosinophil, monocytes, dendritic cells, neutrophils, andmacrophages, including tissue-specific macrophages, for example,alveolar macrophages. It is also expressed in endothelial and epithelialcells in the lung and other tissues, and in muscle cells [see, e.g.,Bielenberg et al., Amer. J. Path. 181:548-559, 2012; Aung, et al., PLoSONE 11(2) e0147358, 2016; Schellenburg et al., Mol. Imm 90:239-244,2017; and Wild et al., Int. J. Exp. Path. 93:81-103, 2012).

Neuropilin-2 also plays a key role in endosome development, for example,by regulating late endosomal maturation, an important aspect ofphagocytosis and efferocytosis, which respectively contribute toclearance of infections and apoptotic cells (See, e.g., Diaz-Vera etal., J. Cell. Sci. 130:697-711, 2017; Dutta et al., Cancer Res.76:418-428, 2016).

Neuropilin-2 is known to be a key player in the pathophysiology of manydiseases (e.g., “NRP2-associated diseases”) and interacts with a broadarray of soluble ligands including semaphorin 3F, VEGF-C and D, andTGF-beta (see, for example, Table N2 and Table N3), and an array ofcellular receptors and co-factors (see, for example, FIGS. 1A-1B andFIG. 2). NRP2 is also polysialated on dendritic cells, and activelyinteracts with the chemokine CCL21 to mediate immune cell migration, andfor which single nucleotide polymorphisms associated with ILD and RAhave been described (see, e.g., Rey-Gallardo et al., Glycobiology20:1139-1146, 2010; Stahl et al., Nat. Genet. 42:508-514, 2013; andMiller et al., Arthritis Rheum. 65:3239-3247). Additionally, soluble,circulating forms of NRP-2 are known (see, e.g., Parker et al.,Structure 23(4) 677-687, 2015), and internal studies have confirmed theexistence of circulating complexes of HRS polypeptides and NRP-2polypeptides in circulation. Accordingly, given the central role playedby NRP2 in pathophysiology in a broad range of diseases, it is evidentthat interactions between NRP2 and NRP2 ligand(s) (for example, NRP2ligands from Table N2 and Table N3), and the modulation of thoseinteractions with antibodies against NRP2 to selectively change thecorresponding biological activities, provides broad potential for thetreatment of diseases, including NRP2 associated diseases.

NRP2 is a single transmembrane receptor with a predominant extracellularregion containing two CUB domains (A1/A2 combined domain), two FactorV/VIII homology domains (B1/B2 combined domain), and a MAM domain (Cdomain) (see FIGS. 1A-1B). The A1A2 combined domain interacts with semaregion of the semaphorins, and the B1 domain interacts with thesemaphorin PSI and Ig-like domains. NRP2 has a higher affinity forSEMA3F and 3G; in contrast, SEMAs 3A, 3B and 3E preferentially interactwith NRP1. Both NRP1 and NRP2 have similar affinity for SEMA 3C. TheB1B2 combined domain interacts with several growth factors containingheparin-binding domains, including VEGF C & D, placenta growth factor(PIGF)-2, fibroblast growth factor (FGF), galectin, hepatocyte growthfactor (HGF), platelet derived growth factor (PDGF), and transforminggrowth factor (TGF)-beta (see, for example, Prud'homme et al.,Oncotarget. 3:921-939, 2012). NRP2 also interacts with various growthfactor-specific receptors, and interactions with these receptors occurindependently of binding to SEMAs. In this context, integrins and growthfactor receptors like VEGF receptor, TGF-beta receptor, c-Met, EGFR,FGFR, PDGFR, have been shown to interact with NRPs and in general appearto increase the affinity of each ligand for its receptor and to modulatedown stream signaling. The C domain (Mam) domain does not appear to berequired for ligand binding, but appears essential for signaling

Accordingly, anti-NRP2 antibodies that bind to the A1 and/or A2 domainsof NRP2 have the potential to selectively modulate semaphorin binding.Likewise, anti-NRP2 antibodies that bind to the B1 domain have thepotential to modulating both semaphorin and VEGF and growth factorbinding, and anti-NRP2 antibodies that bind to the B2 domain have thepotential to selectively modulate VEGF and growth factor binding.Antibodies that bind to the C domain might not directly impact NRP2ligand binding, but have the potential to modulate NRP2 downstreamsignaling. Additional diversity in the functional effects of specificanti-NRP2 antibodies may be expected based on their binding mode, and asa result of steric effects, which may indirectly impact ligand binding.

NRP2 can form homodimers as well as heterodimers, and is heavilyglycosylated. NRP2 has different splice variants which are between about551 and 926 amino acids long. Two major variants for NRP2 arecategorized as NRP2a and NRP2b. These differ in their intracellular Cterminal part (FIGS. 1A-1B) in which for NRP2a, the c-terminal domaincomprises 42 amino acids and a PDZ-binding domain with the C-terminalSEA amino acid sequence. By contrast, NRP2b comprises a 46 amino acid Cterminal domain which shares about 11% of the intracellular andtransmembrane sequence of NRP2a. Between the MAM domain and thetransmembrane domain, additional splicing can occur and 5 additionalamino acids (GENFK) can be added to either the NRP2a, or NRP2bforms—these variants are named based on the number additional aminoacids added through alternative splicing. Thus the two additionalvariants of NRP2 are named NRP2a(17) and NRP2a(22) and the two differenttransmembrane variants for NRP2b are named NRP2b(0) and NRP2b(5).Additionally, a soluble form called sNRP2b can be generated. ExemplaryNRP2 polypeptide sequences are provided in Table N1 below.

TABLE N1 Exemplary Human NRP2 Polypeptides SEQ ID Name Residues SequenceNO: Human full   1-931 MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYITS   1length PGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEKHDCK NRP2YDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIRFTS Variant 1DYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEK NRP2a(22)YPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGENFKVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMSSLGVLLGATCAGLLLYCTCSYSGLSSRSCTTLENYNFELYDGLKHKVKMNH QKCCSEA Human NRP2   1-926MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYITS   2 Variant 2PGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEKHDCK precursorYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIKFTS NRP2a(17)DYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMSSLGVLLGATCAGLLLYCTCSYSGLSSRSCTTLENYNFELYDGLKHKVKMNHQKCCS EA Human NRP2   1-909MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYITS   3 Variant 3PGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEKHDCK precursorYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIKFTS NRP2a(0)DYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFADEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMSSLGVLLGATCAGLLLYCTCSYSGLSSRSCTT LENYNFELYDGLKHKVKMNHQKCCSEAHuman NRP2   1-906 MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYITS   4Variant 4 PGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEKHDCK precursorYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIKFTS NRP2b(5)DYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGENFKGGTLLPGTEPTVDTVPMQPIPAYWYYVMAAGGAVLVLVSVALALVLHYHRFRYAAKKTDHSITYKTSHY TNGAPLAVEPTLTIKLEQDRGSHCHuman NRP2   1-901 MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYI   5Variant 5 TSPGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEK precursorHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYI NRP2b(0)KFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGGTLLPGTEPTVDTVPMQPIPAYWYYVMAAGGAVLVLVSVALALVLHYHRFRYAAKKTDHSITYKTSHYT NGAPLAVEPTLTIKLEQDRGSHCHuman NRP2   1-555 MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYITS   6Variant 6 PGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEKHDCK precursorYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIKFTS S9NRP2bDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEK SolubleYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKY NRP2DWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQP KVGCSWRPL Human NRP2  23-926QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP   7 Variant 2NQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG NRP2a(17)NIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMSSLGVLLGATCAGLLLYCTCSYSGLSSRSCTTLENYN FELYDGLKHKVKMNHQKCCSEA NRP2 23-901 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP   8 spliceNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG variant 5NIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSED NRP2b(0)CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGGTLLPGTEPTVDTVPMQPIPAYWYYVMAAGGAVLVLVSVALALVLHYHRFRYAAKKTDHSITYKTSHYTNGAPLAVEPTLTIKLEQDRGSHC Soluble  23-555QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP   9 NRP2NQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG S9Nrp-2bNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKV SYSLNGKDWEYIQDPRTQQPKVGCSWRPLNRP2 A1  28-141 CGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEPNQKIV  10 domainLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEI NRP2 A2 149-265CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQ  11 domainFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYY NRP2 B1 280-426PLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPN  12 domainLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTR FVRIRPQTWHSGIALRLELFG NRP2 B2438-591 LGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIP  13 domainQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRF DPIPAQYVRVYPERWSPAGIGMRLEVLGNRP2 C 641-794 PSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPD  14 domainDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDM EYQIVFEGVIGKGRSGEIAIDDIRISTDNRP2 23-265 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP  15 A1A2NQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG combinedNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSED domainsCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYY NRP2 149-426CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQ  16 A2B1FLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGT combinedKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLEN domainsFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHA PLLTRFVRIRPQTWHSGIALRLELFGNRP2  23-426 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP  17 A1A2B1NQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG combinedNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSED domainsCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHA PLLTRFVRIRPQTWHSGIALRLELFGNRP2  23-595 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP  18 A1A2B1B2NQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG combinedNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSED domainsCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPA QYVRVYPERWSPAGIGMRLEVLGCDWTNRP2 145-595 GSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKME  19 A2B1B2IILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGK combinedYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQE domainsPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWT NRP2 276-595QCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNG  20 B1B2WTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVK combinedSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAP domainsLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQ YVRVYPERWSPAGIGMRLEVLGCDWTNRP2 v2-  23-855 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP  21Fc fusion NQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG proteinNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK NRP2 145-595GSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKME  22 A2B1B2-FcIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK NRP2 438-794LGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIP  89 B2CQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFV combinedRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRF domainsDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFE GVIGKGRSGEIAIDDIRISTD NRP2276-794 QCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNG  90 B1B2CWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVK combinedSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAP domainsLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKG RSGEIAIDDIRISTD NRP2 149-802CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQ 121 A2B1B2CFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGT combinedKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLEN domainsFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGK GRSGEIAIDDIRISTDVPLENCME NRP223-802 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP 122 A1A2B1B2CNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG combinedNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSED domainsCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGK GRSGEIAIDDIRISTDVPLENCMENRP2A QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP 123 A1A2B1B2CNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG combinedNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSED domains +CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQ juxta-FLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGT membraneKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDP NRP2BQPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP 124 A1A2B1B2CNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG combinedNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSED domains +CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQ juxta-FLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGT membraneKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGGTLLPGTEPTV DTVPMQPIPAY NRP2QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEP 120 (23-595)-NQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCG FcNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSISISP GK

In certain embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to a full-length human NRP2polypeptide or a human NRP2 polypeptide selected from Table N1. In someembodiments, the antibody or antigen-binding fragment thereof binds tothe human NRP2 polypeptide with an affinity of about 10 pM to about 500pM or to about 50 nM, or about, at least about, or no more than about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM,25 nM, or 50 nM, or optionally with an affinity that ranges from about10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM toabout 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM,about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pMto about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, orabout 25 nM to about 50 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof that specifically binds to at least one epitope in atleast one neuropilin domain. Exemplary neuropilin domains include one ormore of the neuropilin A1 domain (SEQ ID NO:10), neuropilin A2 domain(SEQ ID NO:11), neuropilin B1 domain (SEQ ID NO:12), neuropilin B2domain (SEQ ID NO:13), neuropilin C domain (SEQ ID NO:14), neuropilinA1/A2 combined domain (SEQ ID NO:15), neuropilin B1/B2 combined domain(SEQ ID NO:20), neuropilin A2/B1 combined domain (SEQ ID NO:16),neuropilin B2/C combined domain (SEQ ID NO:89), neuropilin A2/B1/B2combined domain (SEQ ID NO:19), neuropilin A2/B1/B2/C combined domain(SEQ ID NO:121), neuropilin A1/A2/B1 combined domain (SEQ ID NO:17),neuropilin A1/A2/B1/B2 combined domain (SEQ ID NO:18), neuropilinA1/A2/B1/B2/C combined domain (SEQ ID NO:122), and the neuropilinB1/B2/C combined domain (SEQ ID NO: 90). In specific embodiments, the atleast one antibody or antigen-binding fragment thereof specificallybinds to at least one epitope in the neuropilin 131 domain, theneuropilin B2 domain, and/or the neuropilin B1/B2 combined domain (seeTable N1). In particular embodiments, the antibody or antigen-bindingfragment thereof binds to the at least one domain (or at least oneepitope therein) with an affinity of about 10 pM to about 500 pM or toabout 50 nM, or about, at least about, or no more than about 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or50 nM, or optionally with an affinity that ranges from about 10 pM toabout 500 pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM,about 10 pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM toabout 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 400pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about50 pM, or about 20 pM to about 200 pM, about 30 pM to about 300 pM,about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM toabout 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM,about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM toabout 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25nM to about 50 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin A1 domain, the neuropilin A2 domain, and/or the neuropilinA1A2 combined domain, including adjacent linker regions, for example, atabout residues (neuropilin A1 domain) 20-148, 30-141, 40-141, 50-141,60-141, 70-141, 80-141, 90-141, 100-141, 110-141, 120-141, 130-141;20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50,20-40, or 20-30 as defined by SEQ ID NO:1 (FL human NRP2); or, forexample, at about residues (neuropilin A2 domain) 142-280, 150-265,160-265, 170-265, 180-265, 190-265, 200-265, 210-265, 220-265, 230-265,240-265, 250-265, 260-265, 141-270, 141-260, 141-250, 141-240, 141-230,141-220, 141-210, 141-200, 141-190, 141-180, 141-170, 141-160, 141-150,200-250, 210-250, 220-250, 230-250, 200-240, 210-240, 220-240, 230-240,227-247, 228-247, 229-247, 230-247, 231-247, 232-247, 233-247, 234-247,235-247, 236-247; 227-246, 227-245, 227-244, 227-243, 227-242, 227-241,227-240, 227-239, 227-238; 235-240, 236-239, 236-238, or residue 237 asdefined by SEQ ID NO:1 (FL human NRP2); or, for example, at aboutresidues (combined A1A2 domain) 20-280, 30-280, 40-280, 50-280, 60-280,70-280, 80-280, 90-280, 100-280, 110-280, 120-280, 130-280, 140-280,150-280, 160-280, 170-280, 180-280, 190-280, 200-280, 210-280, 220-280,230-280, 240-280, 260-280, 270-280, 20-270, 20-260, 20-250, 20-240,20-230, 20-220, 20-210, 20-200, 20-190, 20-180, 20-170, 20-160, 20-150,20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60,20-50, 20-40, or 20-30 as defined by SEQ ID NO:1 (FL human NRP2).

In particular embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin B1 domain (SEQ ID NO:12), the neuropilin B2 domain (SEQ IDNO:13), and/or the neuropilin B1/B2 combined domain (SEQ ID NO:20),including adjacent linker regions, for example, at about residues(neuropilin B1 domain) 266-426, 280-426, 290-426, 300-426, 310-426,320-426, 330-426, 340-426, 350-426, 360-426, 370-426, 380-426, 390-426,400-426, 410-426, 420-426, 280-420, 280-410, 280-400, 280-390, 280-380,280-370, 280-360, 280-350, 280-340, 280-330, 280-320, 280-310, 280-300,or 280-290 as defined by SEQ ID NO:1 (FL human NRP2); (neuropilin B2domain) 438-591, 450-591, 460-591, 470-591, 480-591, 490-591, 500-591,510-591, 520-591, 530-591, 540-591, 550-591, 560-591, 570-591, 580-591,438-590, 438-580, 438-570, 438-560, 438-550, 438-540, 438-530, 438-520,438-510, 438-500, 438-490, 438-480, 438-470, 438-460, 438-450 as definedby SEQ ID NO:1 (FL human NRP2); or (neuropilin B1/B2 combined domain)266-591, 276-591, 286-591, 296-591, 306-591, 316-591, 326-591, 336-591,346-591, 356-591, 366-591, 376-591, 386-591, 396-591, 406-591, 416-591,426-591, 436-591, 446-591, 456-591, 466-591, 476-591, 486-591, 498-591,508-591, 518-591, 528-591, 538-591, 548-591, 558-591, 568-591, 578-591,588-591, 266-581, 266-571, 266-561, 266-551, 266-541, 266-531, 266-521,266-511, 266-501, 266-491, 266-481, 266-471, 266-461, 266-451, 266-441,266-431, 266-421, 266-411, 266-401, 266-391, 266-381, 266-371, 266-361,266-351, 266-341, 266-331, 266-321, 266-311, 266-301, 266-291, 266-281,or 266-271 as defined by SEQ ID NO:1 (FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin A2/B1 combined domain and/or the neuropilin B2C combineddomain, including adjacent linker regions, for example, at aboutresidues (neuropilin A2B1 combined domain) 149-437, 159-426, 169-426,179-426, 189-426, 199-426, 209-426, 219-426, 229-426,239-426, 249-426,259-426, 269-426, 279-426, 289-426, 299-426, 309-426, 319-426, 329-426,339-426, 349-426, 359-426, 369-426, 379-426, 389-426, 399-426, 409-426,419-426, 149-436, 149-426, 149-416, 149-406, 149-396, 149-386, 149-376,149-366, 149-356, 149-346, 149-336, 149-326, 149-316, 149-306, 149-296,149-286, 149-276, 149-266, 149-256, 149-246, 149-236, 149-226, 149-216,149-206, 149-196, 146-186, 146-176, 146-166, or 146-155 as defined bySEQ ID NO:1 (FL human NRP2); or, for example, at about residues(neuropilin B2C combined domain) 438-794, 448-794, 458-794, 468-794,478-794, 487-794, 497-794, 507-794, 517-794, 527-794, 537-794, 547-794,557-794, 567-794, 587-794, 597-794, 607-794, 617-794, 627-794, 637-794,647-794, 657-794, 667-794, 677-794, 687-794, 697-794, 707-794, 717-794,727-794, 737-794, 747-794, 757-794, 767-794, 777-794, 787-794, 427-794,438-784, 438-774, 438-764, 438-754, 438-744, 438-734, 438-728, 438-714,438-704, 438-694, 438-684, 438-674, 438-664, 438-654, 438-644, 438-634,438-624, 438-614, 438-604, 438-596, 438-586, 438-576, 438-566, 438-556,438-546, 438-536, 438-526, 438-516, 438-506, 438-494, 438-484, 438-474,438-464, 438-454, 438-444 as defined by SEQ ID NO:1 (FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin C domain, including adjacent linker regions, for example, atabout residues 591-794, 600-794, 610-794, 620-794, 630-794, 640-794,650-794, 660-794, 670-794, 680-794, 690-794, 700-794, 710-794, 720-794,730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 591-790,591-780, 591-770, 591-760, 591-750, 591-740, 591-730, 591-720, 591-710,591-700, 591-690, 591-680, 591-670, 591-660, 591-650, 591-640, 591-630,591-620, 591-610, or 591-600 as defined by SEQ ID NO:1 (FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin B1/B2/C combined domain, including adjacent linker regions,for example, at about residues 276-794, 286-794, 296-794, 306-794,316-794, 326-794, 336-794, 346-794, 356-794, 366-794, 376-794, 387-794,396-794, 406-794, 416-794, 426-794, 436-794, 446-794, 456-794, 466-794,476-794, 486-794, 496-794, 506-794, 516-794, 526-794, 536-794, 546-794,556-794, 566-794, 576-794, 586-794, 596-794, 606-794, 616-794, 626-794,636-794, 646-794, 656-794, 666-794, 676-794, 686-794, 696-794, 706-794,716-794, 726-794, 736-794, 746-794, 756-794, 766-794, 776-794, 786-794,266-794, 276-784, 276-774, 276-764, 276-754, 276-744, 276-734, 276-724,276-714, 276-704, 276-694, 276-684, 276-674, 276-664, 276-654, 276-644,276-634, 276-624, 276-614, 276-604, 276-594, 276-584, 276-574, 276-564,276-554, 276-544, 276-534, 276-524, 276-514, 276-504, 276-594, 276-584,276-574, 276-564, 276-554, 276-544, 276-534, 276-524, 276-514, 276-504,or 276-496 as defined by SEQ ID NO:1 (FL human NRP2).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to a conformational epitope composedof two or more discontinuous epitope regions. In some embodiments, theat least one antibody or antigen-binding fragment thereof specificallybinds to a conformational epitope comprising or consisting of:

(a) a first epitope region within the A1 domain, and second epitoperegion within the A2 domain of the human NPR2 polypeptide;

(b) a first epitope region within the A1 domain, and second epitoperegion within the B1 domain of the human NPR2 polypeptide;

(c) a first epitope region within the A1 domain, and second epitoperegion within the B2 domain of the human NPR2 polypeptide;

(d) a first epitope region within the A1 domain, and second epitoperegion within the C domain of the human NPR2 polypeptide;

(e) a first epitope region within the A2 domain, and second epitoperegion within the B1 domain of the human NPR2 polypeptide;

(f) a first epitope region within the A2 domain, and second epitoperegion within the B2 domain of the human NPR2 polypeptide;

(g) a first epitope region within the A2 domain, and second epitoperegion within the C domain of the human NPR2 polypeptide;

(h) a first epitope region within the B1 domain, and second epitoperegion within the B2 domain of the human NPR2 polypeptide;

(i) a first epitope region within the 131 domain, and second epitoperegion within the C domain of the human NPR2 polypeptide; or

(j) a first epitope region within the B2 domain, and second epitoperegion within the C domain of the human NPR2 polypeptide.

In some embodiments, the antibody or antigen-binding fragment thereofspecifically binds to at least one epitope within a region of a humanNRP2 polypeptide that binds to or interacts with at least one “NRP2ligand”, including any molecule that interacts with or binds reversiblyto human NRP2. General examples of “NRP2 ligands” include polypeptidessuch as HRS polypeptides, soluble ligands, receptors (e.g., cell surfacereceptors), including growth factors, growth factor receptors, andothers, and specific examples of NRP2 ligands are detailed herein. Insome embodiments, the at least one antibody or antigen-binding fragmentthereof modulates (e.g., antagonizes, interferes with, agonizes,enhances) binding of the human NRP2 polypeptide to at least one “NRP2ligand”.

As noted above, in certain embodiments the at least one NRP2 ligand isan HRS polypeptide. Thus, in certain embodiments, an antibody orantigen-binding fragment thereof specifically binds to at least oneepitope within a region of a human NRP2 polypeptide that binds to orinteracts with at least one human HRS polypeptide, and thereby modulatesbinding of the human NRP2 polypeptide to the human HRS polypeptide.Exemplary HRS polypeptides are provided in Table H1 below.

TABLE H1 Exemplary Human HRS polypeptides SEQ ID Name Residues SequenceNO: FL   1-509 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL  91 cytosolicKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIR wild typeCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRRE DLVEEIKRRTGQPLCIC HisRS1^(N1)  1-141 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL  92KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGE LLSLRYDLTVPFARYLAM HisRS1^(N2)  1-408 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL  93KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTE HisRS1^(N3)   1-113MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL  94KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKL HisRS1^(N4)   1-60MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL  95 KAQLGPDESKQKFVLKTPKHisRS1^(N5)   1-243 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL  9627 aa KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVGY PWWNSCSRILNYPKTSRPWRAWETHisRS1^(C1) 405-509 RTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKN  97PKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREE VDVRREDLVEEIKRRTGQPLCICHisRS1^(C2)   1-60 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL  98175-509 KAQLGPDESKQKFVLKTPKDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTS REEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C3)   1-60 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL  99211-509 KAQLGPDESKQKFVLKTPKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPLCIC HisRS1^(C4)   1-100 +MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 99 211-509KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLR SVTSREEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C5)   1-174 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 100211-509 KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEV DVRREDLVEEIKRRTGQPLCICHisRS1^(C6)   1-60 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 101101-509 KAQLGPDESKQKFVLKTPKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRR EDLVEEIKRRTGQPLCIC HisRS1^(C7)  1-100 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 102 175-509KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSR EEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C8) 1-60 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 103 399-509KAQLGPDESKQKFVLKTPKALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRT GQPLCIC HisRS1^(C9) 1-100 +MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 104 399-509KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTG QPLCIC HisRS1^(C10) 369-509MFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTET 105QVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRR EDLVEEIKRRTGQPLCIC HisRS1^(I1)191-333 CLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKF 106RTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFG IDDKISFDLSLARGLDYYTG FL mito.  1-506 MPLLGLLPRRAWASLLSQLLRPPCASCTGAVRCQSQVAEAV 107 wild typeLTSQLKAHQEKPNFIIKTPKGTRDLSPQHMVVREKILDLVISCFKRHGAKGMDTPAFELKETLTEKYGEDSGLMYDLKDQGGELLSLRYDLTVPFARYLAMNKVKKMKRYHVGKVWRRESPTIVQGRYREFCQCDFDIAGQFDPMIPDAECLKIMCEILSGLQLGDFLIKVNDRRIVDGMFAVCGVPESKFRAICSSIDKLDKMAWKDVRHEMVVKKGLAPEVADRIGDYVQCHGGVSLVEQMFQDPRLSQNKQALEGLGDLKLLFEYLTLFGIADKISFDLSLARGLDYYTGVIYEAVLLQTPTQAGEEPLNVGSVAAGGRYDGLVGMFDPKGHKVPCVGLSIGVERIFYIVEQRMKTKGEKVRTTETQVFVATPQKNFLQERLKLIAELWDSGIKAEMLYKNNPKLLTQLHYCESTGIPLVVIIGEQELKEGVIKIRSVASREEVAIKR ENFVAEIQKRLSES 152-398HVGKVWRRESPTIVQGRYREFCQCDFDIAGQFDPMIPDAEC 108LKIMCEILSGLQLGDFLIKVNDRRIVDGMFAVCGVPESKFRAICSSIDKLDKMAWKDVRHEMVVKKGLAPEVADRIGDYVQCHGGVSLVEQMFQDPRLSQNKQALEGLGDLKLLFEYLTLFGIADKISFDLSLARGLDYYTGVIYEAVLLQTPTQAGEEPLNVGSVAAGGRYDGLVGMFDPKGHKVPCVGLSIGVERIFYIVEQR M 294-372QALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGV 109IYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDP Amino-  54-509FVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDT 110 acylationPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPF domain andARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDF anticodonDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRIL bindingDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKG domainLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQ PLCIC Amino-  54-398FVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDT 111 acylationPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPF domainARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVG LSIGVERIFSIVEQRLE Amino- 61-398 GTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKE 112 acylationTLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMN (core)KLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFD domainPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVER IFSIVEQRLE Anticodon 399-509ALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAE 113 bindingLLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRS domainVTSREEVDVRREDLVEEIKRRTGQPLCIC Anticodon 406-501TTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNP 114 bindingKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEV (core) DVRREDLVEEIKRR domainHRS WHEP X_(A)-L-X_(B)-Q-G-X-X-V-R-X-L-K-X-X-K-A-X_(C)-V-X- 115consensus X-L-L-X-L-K-X_(D) Where: X is any amino acidX_(A) is 0-50 amino acids X_(B) is about 5-7 amino acids,preferably 6 amino acids X_(C) is about 7-9 amino acids,preferably 8 amino acids X_(D) is 0-50 amino acids

Thus, in certain embodiments, the at least one NRP2 ligand is selectedfrom Table H1., and the anti-NRP2 antibody or antigen-binding fragmentthereof modulates (e.g., interferes with) binding of a human NRP2polypeptide (for example, a human NRP2 polypeptide selected from TableN1) to a human HRS polypeptide selected from Table H1. In someembodiments, the anti-NRP2 antibody or antigen-binding fragmentspecifically binds to an HRS polypeptide-interacting region of the NRP2polypeptide, and in some instances mimics one or more signalingactivities of the HRS polypeptide binding to the NRP2 polypeptide, forexample, as an agonist antibody. An “HRS polypeptide-interacting region”includes a region or domain of a human NRP2 polypeptide that interactswith a region or domain of human HRS polypeptide, for example, at aligand binding site for a different NRP2 ligand (examples of which areprovided herein), a dimerization domain, a protein-protein interactiondomain, or at a site which is allosterically sensitive within a NRP2polypeptide to modulate the activity of the NRP2 polypeptide.

In certain embodiments, an antibody or antigen-binding fragment thereofis a “blocking antibody”, which fully or substantially inhibits thebinding between a human NRP2 polypeptide (selected, for example, fromTable N1) and an NRP2 ligand such as a human HRS polypeptide (selected,for example, from Table H1) or other NRP2 ligand (for example, selectedfrom Table N2 or Table N3). In some embodiments, a “blocking antibody”inhibits about or at least about 80-100% (e.g., 80, 85, 90, 95, or 100%)of the theoretical maximal binding between the NRP2 polypeptide and theNRP2 ligand (for example, HRS polypeptide) after pre-incubation of the“blocking antibody” with the NRP2 polypeptide in a substantiallystoichiometrically equivalent amount. As used herein, a“stoichiometrically equivalent amount” refers to a situation where thenumber of moles of one substance (e.g., anti-NRP2 antibody) isequivalent or substantially equivalent to the number of moles at leastone other substance (e.g., NRP2 polypeptide) in a given equation orreaction.

In certain embodiments, an antibody or antigen-binding fragment thereofis a “partial-blocking antibody”, which at least partially but not fullyinhibits the binding between a human NRP2 polypeptide (selected, forexample, from Table N1) and an NRP2 ligand such as a human HRSpolypeptide (selected, for example, from Table H1) or other NRP2 ligand(for example, selected from Table N2 or Table N3). In some embodiments,a “partial-blocking antibody” inhibits about or at least about 20-80%(e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%) of thetheoretical maximal binding between the NRP2 polypeptide and the NRP2ligand (for example, HRS polypeptide) after pre-incubation of the“partial-blocking antibody” with the NRP2 polypeptide in astoichiometric amount.

In specific embodiments, the at least one antibody or antigen-bindingfragment thereof specifically inhibits or otherwise reduces the bindingbetween a human NRP2 polypeptide and a HRS polypeptide splice variantselected from Table H1, for example, a HRS splice variant selected fromone or more of HisRS^(N1), HisRS^(N2), HisRS^(N3), HisRS^(N4)(SV9),HisRS^(N5), HisRS^(C1), HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5),HisRS^(C6), HisRS^(C7), HisRS^(C8)(SV11), and HisRS^(C9) (SV14).

As noted above, NRP2 interacts with multiple NRP2 ligands other thanHRS, which mediate downstream signaling events. Additional examples ofNRP2 ligands are provided in Table N2 and Table N3 below.

TABLE N2 Exemplary Neuropilin Ligands Ligand NRP1 NRP2 VEGF-A121 +VEGF-A145 + VEGF-A165 + + VEGF-B167 + VEGF-C + + VEGF-D + + VEGF-E +PIGF-2 + + VEGFR +R1 and R2 +R1, R2, R3 Heparin + + SEMA3A + SEMA 3B, C,D, F, G + + Plexins A1, A2, A3, A4, D1 + + GIPC1 + + TGF-β1, β2, β3, andLAP + + TbRI and TbRII + + FGF-1, 2, 4, and 7 + + FGF receptor 1 + +Integrins (see Table N3) + + Fibronectin + Galectin-1 and Galectin + +Receptors Li-CAM + + Glat-1 + HRS polypeptides (see Table + H1)

TABLE N3 Vertebrate integrins as NRP2 ligands Name Synonyms DistributionLigands α₁β₁ VLA-1 Many Collagens, laminins α₂β₁ VLA-2 Many Collagens,laminins α₃β₁ VLA-3 Many Laminin-5 α₄β₁ VLA-4 Hematopoietic cellsFibronectin, VCAM-1 α₅β₁ VLA-5; widespread fibronectin and proteinasesfibronectin receptor α₆β₁ VLA-6; widespread laminins laminin receptorα₇β₁ muscle, glioma laminins α_(L)β₂ LFA-1 T-lymphocytes ICAM-1, ICAM-2α_(M)β₂ Mac-1, CR3 Neutrophils and monocytes Serum proteins, ICAM-1α_(IIb)β₃ Fibrinogen Platelets fibrinogen, fibronectin^([24]) receptor;gpIIbIIIa α_(V)β₁ ocular melanoma; vitronectin; fibrinogen neurologicaltumors α_(V)β₃ vitronectin activated endothelial cells, vitronectin,fibronectin, fibrinogen, receptor melanoma, glioblastoma osteopontin,Cyr61, thyroxine, TETRAC α_(V)β₅ widespread, esp. fibroblasts,vitronectin and adenovirus epithelial cells α_(V)β₆ proliferatingepithelia, esp. fibronectin; TGFβ1 + 3 lung and mammary gland α_(V)β₈neural tissue; peripheral fibronectin; TGFβ1 + 3 nerve α₆β₄ Epithelialcells Laminin

Thus, in certain embodiments, the at least one NRP2 ligand is selectedfrom Table N2 and/or Table N3.

For example, in some aspects, the at least one NRP2 ligand is a VEGF(vascular endothelial growth factor) ligand selected from VEGF-A145,VEGF-A165, VEGF-C, VEGF-D, and PIGF-2. VEGF-VEGFR2/3-NRP2 interactionsare associated with promoting cell migration, cell growth, cellsurvival, and cell attachment, and also with lymphangiogenesis,increasing vascular permeability, activating integrin signaling,promoting vesicular trafficking and internalization, and slowingcellular differentiation. Accordingly anti-NRP2 antibodies whichmodulate VEGF related NRP2 ligands would be expected find utility inmodulating one or more of these pathways.

In certain aspects, the at least one NRP2 ligand is a semaphorinselected from one or more of SEMA-3B, SEMA-3C, SEMA-3D, SEMA-3F, andSEMA-3B, or a plexin receptor selected from one or more of plexins A1,A2, A3, A4, and D1. SEMAs typically antagonize the effects of VEGF-C,through there is a close dynamic interplay between VEGF and Semasignaling pathways. SEMAs typically function in the immune system tocontrol cell movement, cell migration, cell-cell communication, and cellactivation. SEMA Plexin-NRP2 interactions are associated with inhibitingcell migration, inhibiting cell growth, promoting apoptosis, inhibitingcell attachment, inhibiting integrin signaling, promoting cellulardifferentiation, inhibiting lymphangiogenesis, reducing vascularpermeability, promoting microtubule destabilization, mediating thecollapse of actin cytoskeleton & cell contraction including growth conecollapse and actomyosin contraction, and preventing neuronal cellspreading and inhibiting axon outgrowth. Accordingly anti-NRP2antibodies which modulate SEMA-related NRP2 ligands would be expectedfind utility in modulating one or more of these pathways.

In some aspects, the at least one NRP2 ligand is an integrin selectedfrom one or more of αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α6β1 and α6β4.Integrin-NRP2 interactions are generally associated with increased celladhesion, cell growth, cancer growth and invasiveness. Accordinglyanti-NRP2 antibodies which modulate integrin related NRP2 ligands wouldbe expected find utility in modulating one or more of these pathways.

In some aspects, the at least one NRP2 ligand is selected from TGFβ1,TGFβ2, TGFβ3, and their corresponding TGFβ receptors. TGF-β signaling isstrongly involved in the regulation of EMT in cancer, and also infibrosis development (see, for example, Gemmill et al., Sci. Signal. 10eaag0528, 2017). NRP2B expression is preferentially upregulated by TGF-βsignaling in abnormal lungs, and shows little or no expression in normallung. NRP2B expression enhances migration, invasion, metastasis, andtumorsphere formation, and also enhances acquired EGFR inhibitorresistance associated with EMT in cancer cells. Accordingly anti-NRP2antibodies which modulate TGF-β related NRP2 ligands would be expectedfind utility in modulating one or more of these pathways. Thus, incertain embodiments, an anti-NRP2 antibody or antigen-binding fragmentthereof modulates binding/signaling activity between an NRP2 polypeptideand at least one of the NRP2 ligands from Table N2 and/or Table N3, forexample, by specifically binding to an NRP2 ligand-interacting region ofthe NRP2 polypeptide.

In some instances, the at least one antibody or antigen-binding fragmentthereof antagonizes the binding/signaling activity between the NRP2polypeptide and the at least one NRP2 ligand. For example, in someembodiments, the anti-NRP2 antibody antagonizes or reduces about or atleast about 20-100% (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 90, or 100%) of the theoretical maximal binding/signalingbetween the NRP2 polypeptide and the NRP2 ligand after pre-incubation ofthe anti-NRP2 antibody with the NRP2 polypeptide in a substantiallystoichiometrically equivalent amount.

In some instances, the at least one antibody or antigen-binding fragmentthereof agonizes or enhances the binding/signaling activity between theNRP2 polypeptide and the at least one NRP2 ligand. For instance, in someembodiments, the anti-NRP2 antibody agonizes or enhances by about or atleast about 20%-500% (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400 or 500%) the theoretical maximal binding/signalingactivity between the NRP2 polypeptide and the at least one NRP2 ligandafter pre-incubation of the anti-NRP2 antibody with the NRP2 polypeptidein a substantially stoichiometrically equivalent amount.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofsemaphorins to, or via, the NRP2 polypeptide. In some aspects, suchantibodies do not substantially block the interaction of VEGF-C orrelated NRP2 ligands. In some aspects, such antibodies are agonisticantibodies with respect to semaphorin signaling. In some aspects, suchantibodies are antagonistic antibodies with respect to semaphorinsignaling.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofVEGF-C or related NRP2 ligands to, or via, the NRP2 polypeptide. In someaspects, such antibodies do not substantially block the interaction ofsemaphorins. In some embodiments, such antibodies selectively modulateboth the binding of VEGF-C or related NRP2 ligands and semaphorins tothe NRP2 polypeptide. In some embodiments, such antibodies are agonisticantibodies with respect to VEGF-C signaling. In some aspects, suchantibodies are antagonistic antibodies with respect to VEGF-C signaling.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofintegrins or related NRP2 ligands to the NRP2 polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofTGFβ1, TGFβ2, TGFβ3, or their corresponding TGFβ receptors to the NRP2polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling offibroblast growth factor (FGF), galectin, hepatocyte growth factor(HGF), platelet derived growth factor, and/or their correspondingreceptors to the NRP2 polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or a semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and VEGFR3 or VEGF-C.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and a HRS polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or a semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and a HRS polypeptide, and without substantially modulatingthe binding/signaling activity between the NRP2 polypeptide and VEGFR3or VEGF-C.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and VEGR3 without substantially modulating thebinding/signaling activity between the NRP2 polypeptide and a plexinreceptor and/or a semaphorin.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and VEGR3 or VEGF-C without substantially modulatingthe binding/signaling activity between the NRP2 polypeptide and a HRSpolypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor without substantially modulatingthe ligand binding of semaphorin 3 to NRP2.

In some embodiments, the plexin receptor is selected from plexin A1, A2,A3, A4, and D1. In some embodiments, the semaphorin is selected fromsemaphorin 3B, 3C, 3D, 3F, and 3G.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope within the human NRP2A2 domain which comprises at least 5 contiguous amino acids of SEQ IDNO: 11, wherein the at least one antibody or antigen-binding fragmentthereof selectively inhibits receptor dimerization between NRP2 andplexin A1 without substantially inhibiting dimerization between NRP2 andFLT4 (VEGFR3). In some embodiments, the at least one antibody orantigen-binding fragment thereof specifically binds to an epitope withinamino acids 232-242 of human NRP2 SEQ ID NO: 1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope within the human NRP2B1 domain which comprises at least 5 contiguous amino acids of SEQ IDNO: 12, wherein the at least one antibody or antigen-binding fragmentthereof selectively inhibits receptor dimerization between NRP2 and FLT4(VEGFR3) without substantially inhibiting dimerization between NRP2 andplexin A1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope within the human NRP2B2 domain which comprises at least 5 contiguous amino acids of SEQ IDNO: 13, wherein the at least one antibody or antigen-binding fragmentthereof inhibits receptor dimerization between NRP2 and FLT4 (VEGFR3)and inhibits dimerization between NRP2 and plexin A1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope within the human NRP2C domain which comprises at least 5 contiguous amino acids of SEQ ID NO:14, wherein the at least one antibody or antigen-binding fragmentthereof inhibits receptor dimerization between NRP2 and plexin A1 andpartially inhibits dimerization between NRP2 and FLT4 (VEGFR3).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof has an affinity (Kd or EC₅₀) for each of (i) a humanNRP2 polypeptide and (ii) the corresponding region of a cynomolgusmonkey NRP2 polypeptide, wherein the affinity for (i) and (ii) is withinthe range of about 20 pM to about 200 pM, about 30 pM to about 300 pM,about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM toabout 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM,about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 0.4 toabout 1.2 nM, about 0.9 to about 5.5 nM, about 0.9 to about 5 nM, orabout 1 nM to about 10 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof has an affinity (Kd or EC₅₀) for each of (i) a humanNRP2 polypeptide and (ii) the corresponding region of a murine NRP2polypeptide, wherein the affinity for (i) and (ii) is within the rangeof about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pMto about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90pM to about 900 pM, about 100 pM to about 1 nM, or about 1 nM to about10 nM.

In certain embodiments, an antibody or antigen-binding fragment thereofis characterized by or comprises a heavy chain variable region (V_(H))sequence that comprises complementary determining region V_(H)CDR1,V_(H)CDR2, and V_(H)CDR3 sequences, and a light chain variable region(V_(L)) sequence that comprises complementary determining regionV_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequences. Exemplary V_(H),V_(H)CDR1, V_(H)CDR2, V_(H)CDR3, V_(L), V_(L)CDR1, V_(L)CDR2, andV_(L)CDR3 sequences are provided in Table A1 below.

TABLE A1 Exemplary CDR Sequences SEQ ID Description Sequence NO:Clone 17F7 V_(H)CDR1 GYTFTSYWMH 23 Ab/clone aNRP2-1 V_(H)CDR2AIYPGNSDTSYNQQFKGKA 24 Ab/clone aNRP2-1 V_(H)CDR3 RGGGYFDY 25Ab/clone aNRP2-1 V_(L)CDR1 KASQNVGAAVA 26 Ab/clone aNRP2-1 V_(L)CDR2aASNRYT 27 Ab/clone aNRP2-1 V_(L)CDR3 QQYSSYPLLT 28 Ab/clone aNRP2-1Clone 3F2 V_(H)CDR1 GYTFTSYWMH 29 Ab/clone aNRP2-2 V_(H)CDR2VIHPNSASTFYNERFKT 30 Ab/clone aNRP2-2 V_(H)CDR3 PGTVRRSDY 31Ab/clone aNRP2-2 V_(L)CDR1 RSSQNIVHSTGNTYLE 32 Ab/clone aNRP2-2V_(L)CDR2 KVSNRFS 33 Ab/clone aNRP2-2 V_(L)CDR3 FQGSHVPWT 34Ab/clone aNRP2-2 Clone 8E2 V_(H)CDR1 GFNIKDYYIH 35 Ab/clone aNRP2-6V_(H)CDR2 RIDVEDDETKYAPKFQG 36 Ab/clone aNRP2-6 V_(H)CDR3 PIYGSREAWFAY37 Ab/clone aNRP2-6 V_(L)CDR1 TASSSVSSSYLH 38 Ab/clone aNRP2-6 V_(L)CDR2RTSNLAS 39 Ab/clone aNRP2-6 V_(L)CDR3 HQYYRSPPT 40 Ab/clone aNRP2-6Clone 5H11 V_(H)CDR1 GFNIKDYYIH 41 Ab/clone aNRP2-7 V_(H)CDR2RIDVEDDETKYAPKFQG 42 Ab/clone aNRP2-7 V_(H)CDR3 PIYGSREAFFAY 43Ab/clone aNRP2-7 V_(L)CDR1 TASSSVSSSYLH 44 Ab/clone aNRP2-7 V_(L)CDR2STSNLAS 45 Ab/clone aNRP2-7 V_(L)CDR3 HQYYRSPPT 46 Ab/clone aNRP2-7Clone 7G10 V_(H)CDR1 GFNVKDYYVH 47 Ab/clone aNRP2-8 V_(H)CDR2RIDVEDDETKYAPKFQG 48 Ab/clone aNRP2-8 V_(H)CDR3 PIYGAREAWFAY 49Ab/clone aNRP2-8 V_(L)CDR1 TANSSVSSSYLH 50 Ab/clone aNRP2-8 V_(L)CDR2STSNLAS 51 Ab/clone aNRP2-8 V_(L)CDR3 HQYHRSPPT 52 Ab/clone aNRP2-8Clone 9E7 V_(H)CDR1 GFSLTNYGVY 53 Ab/clone aNRP2-9 V_(H)CDR2VIWSGGSTDYNAAFIS 54 Ab/clone aNRP2-9 V_(H)CDR3 NGPNWDRGYYAMDY 55Ab/clone aNRP2-9 V_(L)CDR1 KSSQSLLNSRNQKNYLA 56 Ab/clone aNRP2-9V_(L)CDR2 FASTRES 57 Ab/clone aNRP2-9 V_(L)CDR3 QQHYSTPFT 58Ab/clone aNRP2-9 Clone 1E3 V_(H)CDR1 GFNIKDYYIH 59 Ab/clone aNRP2-10V_(H)CDR2 RIDVEDDETKYAPKFQG 60 Ab/clone aNRP2-10 V_(H)CDR3 PIYGSREAWFAY61 Ab/clone aNRP2-10 V_(L)CDR1 TASSSVSSSYLH 62 Ab/clone aNRP2-10V_(L)CDR2 STSNLAS 63 Ab/clone aNRP2-10 V_(L)CDR3 HQYYRSPPT 64Ab/clone aNRP2-10 Clone 13D7 V_(H)CDR1 GYTFTSFGIS 65 Ab/clone aNRP2-11V_(H)CDR2 EIYPRSGNTYYNENFKG 66 Ab/clone aNRP2-11 V_(H)CDR3 SSGYYGSTPFPY67 Ab/clone aNRP2-11 V_(L)CDR1 RASQDISNYLN 68 Ab/clone aNRP2-11V_(L)CDR2 YTSRLHS 69 Ab/clone aNRP2-11 V_(L)CDR3 QQGNTLPWT 70Ab/clone aNRP2-11 Clone 20F3 V_(H)CDR1 GYTFTTSGMS 71 Ab/clone aNRP2-12V_(H)CDR2 WINTYSGVPTYADDFKG 72 Ab/clone aNRP2-12 V_(H)CDR3 YYSYYVDFDY 73Ab/clone aNRP2-12 V_(L)CDR1 aASSSVSSSYLY 74 Ab/clone aNRP2-12 V_(L)CDR2STSNLAS 75 Ab/clone aNRP2-12 V_(L)CDR3 HQWSSYPRT 76 Ab/clone aNRP2-12Clone 18E8 V_(H)CDR1 GFSLTSYGVH 77 Ab/clone aNRP2-14 V_(H)CDR2LIWSGGSTDYSPAFIS 78 Ab/clone aNRP2-14 V_(H)CDR3 NSYSSGYYAMDY 79Ab/clone aNRP2-14 V_(L)CDR1 KASQNVGTAVA 80 Ab/clone aNRP2-14 V_(L)CDR2SASNRYT 81 Ab/clone aNRP2-14 V_(L)CDR3 QQYSSYPPYT 82 Ab/clone aNRP2-14Clone 19E8 V_(H)CDR1 GFNIKDSFIH 83 Ab/clone aNRP2-15 V_(H)CDR2RIDPEDDETKYAPKFQG 84 Ab/clone aNRP2-15 V_(H)CDR3 PIYGSREAWFAY 85Ab/clone aNRP2-15 V_(L)CDR1 TASSSVSSSYLH 86 Ab/clone aNRP2-15 V_(L)CDR2RTSNLAS 87 Ab/clone aNRP2-15 V_(L)CDR3 HQYYRSPPT 88 Ab/clone aNRP2-15

Thus, in certain embodiments, an antibody or antigen-binding fragmentthereof comprises

a heavy chain variable region (V_(H)) sequence that comprisescomplementary determining region V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3sequences selected from Table A1 and variants thereof which specificallybind to a human NRP2 polypeptide (selected, for example, from Table N1);and

a light chain variable region (V_(L)) sequence that comprisescomplementary determining region V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences selected from Table A1 and variants thereof which specificallybind to the human NRP2 polypeptide (selected, for example, from TableN1).

In certain embodiments, the CDR sequences are as follows:

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:23-25, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 26-28, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:29-31, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 32-34, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:35-37, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 38-40, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:41-43, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 44-46, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:47-49, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 50-52, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:53-55, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 56-58, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:59-61, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 62-64, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:65-67, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 68-70, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:71-73, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 74-76, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:77-79, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 80-82, respectively, including variantsthereof;

the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:83-85, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 86-88, respectively, including variantsthereof.

Also included are variants thereof, including affinity matured variants,which bind to human NRP2, for example, variants having 1, 2, 3, 4, 5, or6 alterations in one or more of the CDR regions, for example, one ormore the V_(H)CDR1, V_(H)CDR2, V_(H)CDR3, V_(L)CDR1, V_(L)CDR2, and/orV_(L)CDR3 sequences described herein. Exemplary “alterations” includeamino acid substitutions, additions, and deletions.

Merely for illustrative purposes, the binding interactions between ahuman NRP2 polypeptide and an NRP2 ligand can be detected and quantifiedusing a variety of routine methods, including biacore assays (forexample, with appropriately tagged soluble reagents, bound to a sensorchip), FACS analyses with cells expressing a NRP2 polypeptide on thecell surface (either native, or recombinant), immunoassays, fluorescencestaining assays, ELISA assays, and microcalorimetry approaches such asITC (Isothermal Titration Calorimetry).

In certain embodiments, an antibody or antigen-binding fragment thereofcomprises variant or otherwise modified Fc region(s), including thosehaving altered properties or biological activities relative to wild-typeFc region(s). Examples of modified Fc regions include those havingmutated sequences, for instance, by substitution, insertion, deletion,or truncation of one or more amino acids relative to a wild-typesequence, hybrid Fc polypeptides composed of domains from differentimmunoglobulin classes/subclasses, Fc polypeptides having alteredglycosylation/sialylation patterns, and Fc polypeptides that aremodified or derivatized, for example, by biotinylation (see, e.g., USApplication No. 2010/0209424), phosphorylation, sulfation, etc., or anycombination of the foregoing. Such modifications can be employed toalter (e.g., increase, decrease) the binding properties of the Fc regionto one or more particular FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc,FcγRIIIa, FcγRIIIb, FcRn), its pharmacokinetic properties (e.g.,stability or half-life, bioavailability, tissue distribution, volume ofdistribution, concentration, elimination rate constant, eliminationrate, area under the curve (AUC), clearance, C_(max), t_(max), C_(min),fluctuation), its immunogenicity, its complement fixation or activation,and/or the CDC/ADCC/ADCP-related activities of the Fc region, amongother properties described herein, relative to a corresponding wild-typeFc sequence of an antibody or antigen-binding fragment thereof. Includedare modified Fc regions of human and/or mouse origin.

Also included are antibodies or antigen-binding fragments thereof thatcomprise hybrid Fc regions, for example, Fc regions that comprise acombination of Fc domains (e.g., hinge, CH₂, CH₃, CH₄) fromimmunoglobulins of different species (e.g., human, mouse), different Igclasses, and/or different Ig subclasses. General examples include hybridFc regions that comprise, consist of, or consist essentially of thefollowing combination of CH₂/CH₃ domains: IgA1/IgA1, IgA1/IgA2,IgA1/IgD, IgA1/IgE, IgA1/IgG1, IgA1/IgG2, IgA1/IgG3, IgA1/IgG4,IgA1/IgM, IgA2/IgA1, IgA2/IgA2, IgA2/IgD, IgA2/IgE, IgA2/IgG1,IgA2/IgG2, IgA2/IgG3, IgA2/IgG4, IgA2/IgM, IgD/IgA1, IgD/IgA2, IgD/IgD,IgD/IgE, IgD/IgG1, IgD/IgG2, IgD/IgG3, IgD/IgG4, IgD/IgM, IgE/IgA1,IgE/IgA2, IgE/IgD, IgE/IgE, IgE/IgG1, IgE/IgG2, IgE/IgG3, IgE/IgG4,IgE/IgM, IgG1/IgA1, IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG1, IgG1/IgG2,IgG1/IgG3, IgG1/IgG4, IgG1/IgM, IgG2/IgA1, IgG2/IgA2, IgG2/IgD,IgG2/IgE, IgG2/IgG1, IgG2/IgG2, IgG2/IgG3, IgG2/IgG4, IgG2/IgM,IgG3/IgA1, IgG3/IgA2, IgG3/IgD, IgG3/IgE, IgG3/IgG1, IgG3/IgG2,IgG3/IgG3, IgG3/IgG4, IgG3/IgM, IgG4/IgA1, IgG4/IgA2, IgG4/IgD,IgG4/IgE, IgG4/IgG1, IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM,IgM/IgA1, IgM/IgA2, IgM/IgD, IgM/IgE, IgM/IgG1, IgM/IgG2, IgM/IgG3,IgM/IgG4, IgM/IgM (or fragments or variants thereof), and optionallyinclude a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3,or IgG4, and/or a CH₄ domain from IgE and/or IgM. In specificembodiments, the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Additional examples include hybrid Fc regions that comprise, consist of,or consist essentially of the following combination of CH₂/CH₄ domains:IgA1/IgE, IgA2/IgE, IgD/IgE, IgE/IgE, IgG1/IgE, IgG2/IgE, IgG3/IgE,IgG4/IgE, IgM/IgE, IgA1/IgM, IgA2/IgM, IgD/IgM, IgE/IgM, IgG1/IgM,IgG2/IgM, IgG3/IgM, IgG4/IgM, IgM/IgM (or fragments or variantsthereof), and optionally include a hinge from one or more of IgA1, IgA2,IgD, IgG1, IgG2, IgG3, IgG4, and/or a CH₃ domain from one or more ofIgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In specificembodiments, the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Certain examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of CH₃/CH₄ domains:IgA1/IgE, IgA2/IgE, IgD/IgE, IgE/IgE, IgG1/IgE, IgG2/IgE, IgG3/IgE,IgG4/IgE, IgM/IgE, IgA1/IgM, IgA2/IgM, IgD/IgM, IgE/IgM, IgG1/IgM,IgG2/IgM, IgG3/IgM, IgG4/IgM, IgM/IgM (or fragments or variantsthereof), and optionally include a hinge from one or more of IgA1, IgA2,IgD, IgG1, IgG2, IgG3, IgG4, and/or a CH₂ domain from one or more ofIgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In specificembodiments, the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Particular examples include hybrid Fc regions that comprise, consist of,or consist essentially of the following combination of hinge/CH₂domains: IgA1/IgA1, IgA1/IgA2, IgA1/IgD, IgA1/IgE, IgA1/IgG1, IgA1/IgG2,IgA1/IgG3, IgA1/IgG4, IgA1/IgM, IgA2/IgA1, IgA2/IgA2, IgA2/IgD,IgA2/IgE, IgA2/IgG1, IgA2/IgG2, IgA2/IgG3, IgA2/IgG4, IgA2/IgM,IgD/IgA1, IgD/IgA2, IgD/IgD, IgD/IgE, IgD/IgG1, IgD/IgG2, IgD/IgG3,IgD/IgG4, IgD/IgM, IgG1/IgA1, IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG1,IgG1/IgG2, IgG1/IgG3, IgG1/IgG4, IgG1/IgM, IgG2/IgA1, IgG2/IgA2,IgG2/IgD, IgG2/IgE, IgG2/IgG1, IgG2/IgG2, IgG2/IgG3, IgG2/IgG4,IgG2/IgM, IgG3/IgA1, IgG3/IgA2, IgG3/IgD, IgG3/IgE, IgG3/IgG1,IgG3/IgG2, IgG3/IgG3, IgG3/IgG4, IgG3/IgM, IgG4/IgA1, IgG4/IgA2,IgG4/IgD, IgG4/IgE, IgG4/IgG1, IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM(or fragments or variants thereof), and optionally include a CH₃ domainfrom one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, orIgM, and/or a CH₄ domain from IgE and/or IgM. In specific embodiments,the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Certain examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of hinge/CH₃ domains:IgA1/IgA1, IgA1/IgA2, IgA1/IgD, IgA1/IgE, IgA1/IgG1, IgA1/IgG2,IgA1/IgG3, IgA1/IgG4, IgA1/IgM, IgA2/IgA1, IgA2/IgA2, IgA2/IgD,IgA2/IgE, IgA2/IgG1, IgA2/IgG2, IgA2/IgG3, IgA2/IgG4, IgA2/IgM,IgD/IgA1, IgD/IgA2, IgD/IgD, IgD/IgE, IgD/IgG1, IgD/IgG2, IgD/IgG3,IgD/IgG4, IgD/IgM, IgG1/IgA1, IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG1,IgG1/IgG2, IgG1/IgG3, IgG1/IgG4, IgG1/IgM, IgG2/IgA1, IgG2/IgA2,IgG2/IgD, IgG2/IgE, IgG2/IgG1, IgG2/IgG2, IgG2/IgG3, IgG2/IgG4,IgG2/IgM, IgG3/IgA1, IgG3/IgA2, IgG3/IgD, IgG3/IgE, IgG3/IgG1,IgG3/IgG2, IgG3/IgG3, IgG3/IgG4, IgG3/IgM, IgG4/IgA1, IgG4/IgA2,IgG4/IgD, IgG4/IgE, IgG4/IgG1, IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM(or fragments or variants thereof), and optionally include a CH₂ domainfrom one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, orIgM, and/or a CH₄ domain from IgE and/or IgM. In specific embodiments,the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Some examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of hinge/CH₄ domains:IgA1/IgE, IgA1/IgM, IgA2/IgE, IgA2/IgM, IgD/IgE, IgD/IgM, IgG1/IgE,IgG1/IgM, IgG2/IgE, IgG2/IgM, IgG3/IgE, IgG3/IgM, IgG4/IgE, IgG4/IgM (orfragments or variants thereof), and optionally include a CH₂ domain fromone or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM,and/or a CH₃ domain from one or more of IgA1, IgA2, IgD, IgE, IgG1,IgG2, IgG3, IgG4, or IgM.

Specific examples of hybrid Fc regions can be found, for example, in WO2008/147143, which are derived from combinations of IgG subclasses orcombinations of human IgD and IgG.

Also included are antibodies or antigen-binding fragments thereof havingderivatized or otherwise modified Fc regions. In certain aspects, the Fcregion may be modified by phosphorylation, sulfation, acrylation,glycosylation, methylation, farnesylation, acetylation, amidation, andthe like, for instance, relative to a wild-type or naturally-occurringFc region. In certain embodiments, the Fc region may comprise wild-typeor native glycosylation patterns, or alternatively, it may compriseincreased glycosylation relative to a native form, decreasedglycosylation relative to a native form, or it may be entirelydeglycosylated. As one example of a modified Fc glycoform, decreasedglycosylation of an Fc region reduces binding to the C1q region of thefirst complement component C1, a decrease in ADCC-related activity,and/or a decrease in CDC-related activity. Certain embodiments thusemploy a deglycosylated or aglycosylated Fc region. See, e.g., WO2005/047337 for the production of exemplary aglycosylated Fc regions.Another example of an Fc region glycoform can be generated bysubstituting the Q295 position with a cysteine residue (see, e.g., U.S.Application No. 2010/0080794), according to the Kabat et al. numberingsystem. Certain embodiments may include Fc regions where about 80-100%of the glycoprotein in Fc region comprises a mature core carbohydratestructure that lacks fructose (see, e.g., U.S. Application No.2010/0255013). Some embodiments may include Fc regions that areoptimized by substitution or deletion to reduce the level offucosylation, for instance, to increase affinity for FcγRI, FcγRIa, orFcγRIIIa, and/or to improve phagocytosis by FcγRIIa-expressing cells(see U.S. Application Nos. 2010/0249382 and 2007/0148170).

As another example of a modified Fc glycoform, an Fc region of anantibody or antigen-binding fragment thereof may compriseoligomannose-type N-glycans, and optionally have one or more of thefollowing: increased ADCC effector activity, increased binding affinityfor FcγRIIIA (and certain other FcRs), similar or increased bindingspecificity for the target of the NRP2 polypeptide, similar or higherbinding affinity for the target of the NRP2 polypeptide, and/or similaror lower binding affinity for mannose receptor, relative to acorresponding Fc region that contains complex-type N-glycans (see, e.g.,U.S. Application No. 2007/0092521 and U.S. Pat. No. 7,700,321). Asanother example, enhanced affinity of Fc regions for FcγRs has beenachieved using engineered glycoforms generated by expression ofantibodies in engineered or variant cell lines (see, e.g., Umana et al.,Nat Biotechnol. 17:176-180, 1999; Davies et al., Biotechnol Bioeng.74:288-294, 2001; Shields et al., J Biol Chem. 277:26733-26740, 2002;Shinkawa et al., 2003, J Biol Chem. 278:3466-3473, 2003; and U.S.Application No. 2007/0111281). Certain Fc region glycoforms comprise anincreased proportion of N-glycoside bond type complex sugar chains,which do not have the 1-position of fucose bound to the 6-position ofN-acetylglucosamine at the reducing end of the sugar chain (see, e.g.,U.S. Application No. 2010/0092997). Particular embodiments may includeIgG Fc region that is glycosylated with at least one galactose moietyconnected to a respective terminal sialic acid moiety by an α-2,6linkage, optionally where the Fc region has a higher anti-inflammatoryactivity relative to a corresponding, wild-type Fc region (see U.S.Application No. 2008/0206246). Certain of these and related alteredglycosylation approaches have generated substantial enhancements of thecapacity of Fc regions to selectively bind FcRs such as FcγRIII, tomediate ADCC, and to alter other properties of Fc regions, as describedherein.

Certain variant, fragment, hybrid, or otherwise modified Fc regions ofan antibody or antigen-binding fragment thereof may have altered bindingto one or more FcRs, and/or corresponding changes to effector function,relative to a corresponding, wild-type Fc sequence (e.g., same species,same Ig class, same Ig subclass). For instance, such Fc regions may haveincreased binding to one or more of Fcγ receptors, Fcα receptors, Fcεreceptors, and/or the neonatal Fc receptor, relative to a corresponding,wild-type Fc sequence. In other embodiments, variant, fragment, hybrid,or modified Fc regions may have decreased binding to one or more of Fcγreceptors, Fcα receptors, Fcε receptors, and/or the neonatal Fcreceptor, relative to a corresponding, wild-type Fc sequence. SpecificFcRs are described elsewhere herein.

In some embodiments, an antibody comprises an Fc domain, comprising oneor more mutations to increase binding to one or more of Fcγ receptors,Fcα receptors, Fcε receptors, and/or the neonatal Fc receptor, relativeto a corresponding, wild-type Fc sequence. In some embodiments, anantibody comprises an IgG1 or IgG3 Fc domain, comprising one or moremutations to increase binding to one or more of Fcγ receptors, Fcαreceptors, Fcε receptors, and/or the neonatal Fc receptor, relative to acorresponding, wild-type Fc sequence. In some embodiments, an antibodycomprises an Fc domain, comprising one or more mutations to increaseeffector function. In some embodiments the at least one antibodycomprises an Fc domain selected from a human IgG1 and IgG3, comprisingone or more mutations to increase effector function.

In some embodiments, an antibody is blocking antibody that comprises anFc domain with high effector activity. In some embodiments, the blockingantibody comprises an Fc domain selected from a human IgG1 and IgG3,comprising one or more mutations to increase effector function. In someembodiments, an antibody is a partial-blocking antibody that comprisesan Fc domain with high effector activity. In some embodiments, the apartial-blocking antibody comprises an Fc domain selected from a humanIgG1 and IgG3, comprising one or more mutations to increase effectorfunction. In some embodiments, an antibody is a non-blocking antibodythat comprises an Fc domain with high effector activity. In someembodiments, the non-blocking antibody comprises an Fc domain selectedfrom a human IgG1 or IgG3, comprising one or more mutations to increaseeffector function.

In some embodiments, an antibody comprises an Fc domain, comprising oneor more mutations to decrease binding to one or more of Fcγ receptors,Fcα receptors, Fcε receptors, and/or the neonatal Fc receptor, relativeto a corresponding, wild-type Fc sequence. In some embodiments, anantibody comprises an IgG1 or IgG3 Fc domain, comprising one or moremutations to decrease binding to one or more of Fcγ receptors, Fcαreceptors, Fcε receptors, and/or the neonatal Fc receptor, relative to acorresponding, wild-type Fc sequence. In some embodiments, an antibodycomprises an Fc domain, comprising one or more mutations to decreaseeffector function. In some embodiments, an antibody comprises an Fcdomain selected from a human IgG2 and IgG4, comprising one or moremutations to decrease effector function.

In some embodiments, an antibody is a blocking antibody comprising an Fcdomain with low effector activity. In some embodiments, the blockingantibody comprises an Fc domain selected from a human IgG2 and IgG4,comprising one or more mutations to decrease effector function. In someembodiments, an antibody is a partial-blocking antibody comprising an Fcdomain with low effector activity. In some embodiments, thepartial-blocking antibody comprises an Fc domain selected from a humanIgG2 and IgG4, comprising one or more mutations to decrease effectorfunction. In some embodiments, an antibody is a non-blocking antibodycomprising an Fc domain with low effector activity. In some embodiments,the non-blocking antibody comprises an Fc domain selected from a humanIgG2 and IgG4, comprising one or more mutations to decrease effectorfunction.

Specific examples of Fc variants having altered (e.g., increased,decreased) effector function/FcR binding can be found, for example, inU.S. Pat. Nos. 5,624,821 and 7,425,619; U.S. Application Nos.2009/0017023, 2009/0010921, and 2010/0203046; and WO 2000/42072 and WO2004/016750. Certain examples include human Fc regions having a one ormore substitutions at position 298, 333, and/or 334, for example, S298A,E333A, and/or K334A (based on the numbering of the EU index of Kabat etal.), which have been shown to increase binding to the activatingreceptor FcγRIIIa and reduce binding to the inhibitory receptor FcγRIIb.These mutations can be combined to obtain double and triple mutationvariants that have further improvements in binding to FcRs. Certainembodiments include a S298A/E333A/K334A triple mutant, which hasincreased binding to FcγRIIIa, decreased binding to FcγRIIb, andincreased ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604,2001; and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See alsoengineered Fc glycoforms that have increased binding to FcRs, asdisclosed in Umana et al., supra; and U.S. Pat. No. 7,662,925. Someembodiments include Fc regions that comprise one or more substitutionsselected from 434S, 252Y/428L, 252Y/434S, and 428L/434S (see U.S.Application Nos. 2009/0163699 and 20060173170), based on the EU index ofKabat et al.

Certain variant, fragment, hybrid, or modified Fc regions may havealtered effector functions, relative to a corresponding, wild-type Fcsequence. For example, such Fc regions may have increased complementfixation or activation, increased Clq binding affinity, increasedCDC-related activity, increased ADCC-related activity, and/or increasedADCP-related activity, relative to a corresponding, wild-type Fcsequence. In other embodiments, such Fc regions may have decreasedcomplement fixation or activation, decreased Clq binding affinity,decreased CDC-related activity, decreased ADCC-related activity, and/ordecreased ADCP-related activity, relative to a corresponding, wild-typeFc sequence. As merely one illustrative example, an Fc region maycomprise a deletion or substitution in a complement-binding site, suchas a C1q-binding site, and/or a deletion or substitution in an ADCCsite. Examples of such deletions/substitutions are described, forexample, in U.S. Pat. No. 7,030,226. Many Fc effector functions, such asADCC, can be assayed according to routine techniques in the art. (see,e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Usefuleffector cells for such assays includes, but are not limited to, naturalkiller (NK) cells, macrophages, and other peripheral blood mononuclearcells (PBMC). Alternatively, or additionally, certain Fc effectorfunctions may be assessed in vivo, for example, by employing an animalmodel described in Clynes et al. PNAS. 95:652-656, 1998.

Certain variant hybrid, or modified Fc regions may have alteredstability or half-life relative to a corresponding, wild-type Fcsequence. In certain embodiments, such Fc regions may have increasedhalf-life relative to a corresponding, wild-type Fc sequence. In otherembodiments, variant hybrid, or modified Fc regions may have decreasedhalf-life relative to a corresponding, wild-type Fc sequence. Half-lifecan be measured in vitro (e.g., under physiological conditions) or invivo, according to routine techniques in the art, such as radiolabeling,ELISA, or other methods. In vivo measurements of stability or half-lifecan be measured in one or more bodily fluids, including blood, serum,plasma, urine, or cerebrospinal fluid, or a given tissue, such as theliver, kidneys, muscle, central nervous system tissues, bone, etc. Asone example, modifications to an Fc region that alter its ability tobind the FcRn can alter its half-life in vivo. Assays for measuring thein vivo pharmacokinetic properties (e.g., in vivo mean eliminationhalf-life) and non-limiting examples of Fc modifications that alter itsbinding to the FcRn are described, for example, in U.S. Pat. Nos.7,217,797 and 7,732,570; and U.S. Application Nos. US 2010/0143254 and2010/0143254.

Additional non-limiting examples of modifications to alter stability orhalf-life include substitutions/deletions at one or more of amino acidresidues selected from 251-256, 285-290, and 308-314 in the CH₂ domain,and 385-389 and 428-436 in the CH₃ domain, according to the numberingsystem of Kabat et al. See U.S. Application No. 2003/0190311. Specificexamples include substitution with leucine at position 251, substitutionwith tyrosine, tryptophan or phenylalanine at position 252, substitutionwith threonine or serine at position 254, substitution with arginine atposition 255, substitution with glutamine, arginine, serine, threonine,or glutamate at position 256, substitution with threonine at position308, substitution with proline at position 309, substitution with serineat position 311, substitution with aspartate at position 312,substitution with leucine at position 314, substitution with arginine,aspartate or serine at position 385, substitution with threonine orproline at position 386, substitution with arginine or proline atposition 387, substitution with proline, asparagine or serine atposition 389, substitution with methionine or threonine at position 428,substitution with tyrosine or phenylalanine at position 434,substitution with histidine, arginine, lysine or serine at position 433,and/or substitution with histidine, tyrosine, arginine or threonine atposition 436, including any combination thereof. Such modificationsoptionally increase affinity of the Fc region for the FcRn and therebyincrease half-life, relative to a corresponding, wild-type Fc region.

Certain variant hybrid, or modified Fc regions may have alteredsolubility relative to a corresponding, wild-type Fc sequence. Incertain embodiments, such Fc regions may have increased solubilityrelative to a corresponding, wild-type Fc sequence. In otherembodiments, variant hybrid, or modified Fc regions may have decreasedsolubility relative to a corresponding, wild-type Fc sequence.Solubility can be measured, for example, in vitro (e.g., underphysiological conditions) according to routine techniques in the art.Exemplary solubility measurements are described elsewhere herein.

Additional examples of variants include IgG Fc regions havingconservative or non-conservative substitutions (as described elsewhereherein) at one or more of positions 250, 314, or 428 of the heavy chain,or in any combination thereof, such as at positions 250 and 428, or atpositions 250 and 314, or at positions 314 and 428, or at positions 250,314, and 428 (see, e.g., U.S. Application No. 2011/0183412). In specificembodiments, the residue at position 250 is substituted with glutamicacid or glutamine, and/or the residue at position 428 is substitutedwith leucine or phenylalanine. As another illustrative example of an IgGFc variant, any one or more of the amino acid residues at positions 214to 238, 297 to 299, 318 to 322, and/or 327 to 331 may be used as asuitable target for modification (e.g., conservative or non-conservativesubstitution, deletion). In particular embodiments, the IgG Fc variantCH₂ domain contains amino acid substitutions at positions 228, 234, 235,and/or 331 (e.g., human IgG4 with Ser228Pro and Leu235Ala mutations) toattenuate the effector functions of the Fc region (see U.S. Pat. No.7,030,226). Here, the numbering of the residues in the heavy chain isthat of the EU index (see Kabat et al., “Sequences of Proteins ofImmunological Interest,” 5^(th) Ed., National Institutes of Health,Bethesda, Md. (1991)). Certain of these and related embodiments havealtered (e.g., increased, decreased) FcRn binding and/or serumhalf-life, optionally without reduced effector functions such as ADCC orCDC-related activities.

Additional examples include variant Fc regions that comprise one or moreamino acid substitutions at positions 279, 341, 343 or 373 of awild-type Fc region, or any combination thereof (see, e.g., U.S.Application No. 2007/0224188). The wild-type amino acid residues atthese positions for human IgG are valine (279), glycine (341), proline(343) and tyrosine (373). The substation(s) can be conservative ornon-conservative, or can include non-naturally occurring amino acids ormimetics, as described herein. Alone or in combination with thesesubstitutions, certain embodiments may also employ a variant Fc regionthat comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions selected from the following: 235G, 235R, 236F, 236R, 236Y,237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L,245R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W,247Y, 248F, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, 251H, 251I,251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P,254Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V,256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 264S, 265K,265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R,279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R,279S, 279T, 279W, 279Y, 280T, 283F, 283G, 283H, 283I, 283K, 283L, 283M,283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G,292I, 292L, 293S, 293V, 301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L,315P, 315R, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332F, 332G, 332L,332M, 332S, 332V, 332W, 339D, 339E, 339F, 339G, 339H, 339I, 339K, 339L,339M, 339N, 339Q, 339R, 339S, 339W, 339Y, 341D, 341E, 341F, 341H, 341I,341K, 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y,343A, 343D, 343E, 343F, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q,343R, 343S, 343T, 343V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I,373K, 373L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E,376F, 376G, 376H, 376I, 376L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T,376V, 376W, 376Y, 377G, 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D,380N, 380S, 380T, 382D, 382F, 382H, 382I, 382K, 382L, 382M, 382N, 382P,382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H,424M, 424V, 426D, 426L, 427N, 429A, 429F, 429M, 430A, 430D, 430F, 430G,430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V,430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, 438K, 438L, 438T, 438W,439E, 439H, 439Q, 440D, 440E, 440F, 440G, 440H, 440I, 440K, 440L, 440M,440Q, 440T, 440V or 442K. As above, the numbering of the residues in theheavy chain is that of the EU index (see Kabat et al., supra). Suchvariant Fc regions typically confer an altered effector function oraltered serum half-life upon the antibody to which the variant Fc regionis operably attached. Preferably the altered effector function is anincrease in ADCC, a decrease in ADCC, an increase in CDC, a decrease inCDC, an increase in Clq binding affinity, a decrease in Clq bindingaffinity, an increase in FcR (preferably FcRn) binding affinity or adecrease in FcR (preferably FcRn) binding affinity as compared to acorresponding Fc region that lacks such amino acid substitution(s).

Additional examples include variant Fc regions that comprise an aminoacid substitution at one or more of position(s) 221, 222, 224, 227, 228,230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244,245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288,290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318,320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335 336 and/or 428 (see, e.g., U.S. Pat. No. 7,662,925). In specificembodiments, the variant Fc region comprises at least one amino acidsubstitution selected from the group consisting of: P230A, E233D, L234E,L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q,S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V2661, E272Y, K274T,K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D,S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328O, L328D, L328V,L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332O, T335D, T335R,and T335Y. In other specific embodiments, the variant Fc regioncomprises at least one amino acid substitution selected from the groupconsisting of: V264I, F243L/V264I, L328M, I332E, L328M/I332E,V264I/I332E, S298A/I332E, S239E/I332E, S239Q/I332E, S239E, A330Y, I332D,L328I/I332E, L328Q/I332E, V264T, V240I, V2661, S239D, S239D/I332D,S239D/I332E, S239D/I332N, S239D/I332Q, S239E/I332D, S239E/I332N,S239E/I332Q, S239N/I332D, S239N/I332E, S239Q/I332D, A330Y/I332E,V264I/A330Y/I332E, A330L/I332E, V264I/A330L/I332E, L234E, L234Y, L234I,L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T,L328D/I332E, L328V/I332E, L328T/I332E, L328I/I332E, S239E/V264I/I332E,S239Q/V264I/I332E, S239E/V264I/A330Y/I332E, S239D/A330Y/I332E,S239N/A330Y/I332E, S239D/A330L/I332E, S239N/A330L/I332E,V264I/S298A/I332E, S239D/S298A/I332E, S239N/S298A/I332E,S239D/V264I/I332E, S239D/V264I/S298A/I332E, S239D/V264I/A330L/1332E,S239D/I332E/A330I, P230A, P230A/E233D/I332E, E272Y, K274T, K274E, K274R,K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V,K326I, K326T, T335D, T335R, T335Y, V240I/V2661, S239D/A330Y/I332E/L234I,S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I,S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/K326E, andS239D/A330Y/I332E/K326T, In more specific embodiments, the variant Fcregion comprises a series of substitutions selected from the groupconsisting of: N297D/I332E, F241Y/F243Y/V262T/V264T/N297D/I332E,S239D/N297D/I332E, S239E/N297D/I332E, S239D/D265Y/N297D/1332E,S239D/D265H/N297D/1332E, V264E/N297D/I332E, Y296N/N297D/I332E,N297D/A330Y/I332E, S239D/D265V/N297D/I332E, S239D/D265I/N297D/I332E, andN297D/S298A/A330Y/I332E. In specific embodiments, the variant Fc regioncomprises an amino acid substitution at position 332 (using thenumbering of the EU index, Kabat et al., supra). Examples ofsubstitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L,332M, 332N, 332P, 332O, 332R, 332S, 332T, 332V, 332W and 332Y. Thenumbering of the residues in the Fc region is that of the EU index ofKabat et al. Among other properties described herein, such variant Fcregions may have increased affinity for an FcγR, increased stability,and/or increased solubility, relative to a corresponding, wild-type Fcregion.

Further examples include variant Fc regions that comprise one or more ofthe following amino acid substitutions: 224N/Y, 225A, 228L, 230S, 239P,240A, 241L, 243S/L/G/H/I, 244L, 246E, 247L/A, 252T, 254T/P, 258K, 261Y,265V, 266A, 267G/N, 268N, 269K/G, 273A, 276D, 278H, 279M, 280N, 283G,285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A,3061/F, 311R, 312N, 315D/K/S, 320R, 322E, 323A, 324T, 325S, 326E/R,332T, 333D/G, 3351, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S,352A, 354A, 355W, 356G, 358T, 361D/Y, 362L, 364C, 365Q/P, 370R, 372L,377V, 378T, 383N, 389S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R,411I, 412A, 414M, 421S, 4221, 426F/P, 428T, 430K, 431S, 432P, 433P,438L, 439E/R, 440G, 441F, 442T, 445R, 446A, 447E, optionally where thevariant has altered recognition of an Fc ligand and/or altered effectorfunction compared with a parent Fc polypeptide, and wherein thenumbering of the residues is that of the EU index as in Kabat et al.Specific examples of these and related embodiments include variant Fcregions that comprise or consist of the following sets of substitutions:(1) N276D, R2920, V305A, I377V, T394A, V412A and K439E; (2) P244L,K246E, D399G and K409R; (3) 5304G, K320R, 5324T, K326E and M358T; (4)F243S, P247L, D265V, V266A, S383N and T411I; (5) H224N, F243L, T393A andH433P; (6) V240A, S267G, G341E and E356G; (7) M252T, P291L, P352A,R355W, N390D, 5408G, S426F and A431S; (8) P228L, T289A, L3650, N389S and5440G; (9) F241L, V273A, K340Q and L441F; (10) F241L, T299A, I332T andM428T; (11) E269K, Y300H, 0342R, V4221 and G446A; (12) T225A, R301c,5304G, D312N, N315D, L351S and N421S; (13) 5254T, L3061, K326R and0362L; (14) H224Y, P230S, V323A, E333D, K338R and 5364C; (15) T3351,K414M and P445R; (16) T3351 and K414M; (17) P247A, E258K, D280N, K288R,N297D, T299A, K322E, 0342R, S354A and L365P; (18) H268N, V279M, A339T,N361D and S426P; (19) C261Y, K290E, L306F, Q311R, E333G and Q438L; (20)E283G, N315K, E333G, R3440, L365P and S442T; (21) 0347R, N361Y andK439R; (22) S239P, S254P, S267N, H285R, N3155, F372L, A378T, N390D,Y391C, F404S, E430K, L432P and K447E; and (23) E269G, Y278H, N325S andK370R, wherein the numbering of the residues is that of the EU index asin Kabat et al. (see, e.g., U.S. Application No. 2010/0184959).

Variant Fc regions can also have one or more mutated hinge regions, asdescribed, for example, in U.S. Application No. 2003/0118592. Forinstance, one or more cysteines in a hinge region can be deleted orsubstituted with a different amino acid. The mutated hinge region cancomprise no cysteine residues, or it can comprise 1, 2, or 3 fewercysteine residues than a corresponding, wild-type hinge region. In someembodiments, an Fc region having a mutated hinge region of this typeexhibits a reduced ability to dimerize, relative to a wild-type Ig hingeregion.

In particular embodiments, the Fc region comprises, consists, orconsists essentially of the Fc from human IgG1 or IgG4 (see, e.g.,Allberse and Schuurman, Immunology. 105:9-19, 2002), or a fragment orvariant thereof. Table F1 below provides exemplary sequences (CH1, hinge(underlined), CH2, and CH3 regions) from human IgG1 and IgG4. Examplesof variant IgG4 sequences that can be employed are described, forexample, in Peters et al., JBC. 287:24525-24533, 2012, and includesubstitutions at C227, C230, C127 (e.g., C127S), and C131 (e.g., C131S).Other variants that can be used include a L445P substitution in IgG4(denoted as IgG4-2) or a D356E and L358M substitution in IgG1, (denotedas IgG1m(zf)).

TABLE F1 Exemplary IgG4 Fc Sequences SEQ ID Name Sequence NO: Wild-typeASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV 116 IgG4HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK S241PASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV 117HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG1m(za)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV 118 GenBank:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP AH007035.2KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG 119 Km3NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

As noted above, antibodies having altered Fc regions typically havealtered (e.g., improved, increased, decreased) pharmacokineticproperties relative to corresponding wild-type Fc region. Examples ofpharmacokinetic properties include stability or half-life,bioavailability (the fraction of a drug that is absorbed), tissuedistribution, volume of distribution (apparent volume in which a drug isdistributed immediately after it has been injected intravenously andequilibrated between plasma and the surrounding tissues), concentration(initial or steady-state concentration of drug in plasma), eliminationrate constant (rate at which drugs are removed from the body),elimination rate (rate of infusion required to balance elimination),area under the curve (AUC or exposure; integral of theconcentration-time curve, after a single dose or in steady state),clearance (volume of plasma cleared of the drug per unit time), C_(max)(peak plasma concentration of a drug after oral administration), t_(max)(time to reach C_(max)), C_(min) (lowest concentration that a drugreaches before the next dose is administered), and fluctuation (peaktrough fluctuation within one dosing interval at steady state).

In particular embodiments, an antibody or antigen-binding fragmentthereof has a biological half life at about pH 7.4, at about aphysiological pH, at about 25° C. or room temperature, and/or at about37° C. or human body temperature (e.g., in vivo, in serum, in a giventissue, in a given species such as rat, mouse, monkey, or human), ofabout or at least about 30 minutes, about 1 hour, about 2 hour, about 3hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours,about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours,about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90hours, about 96 hours, about 120 hours, or about 144 hours or more, orabout 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, orabout 5 weeks, or about 6 weeks or more, or any intervening half-life,including all ranges in between.

In some embodiments, an antibody or antigen-binding fragment thereof hasa T_(m) of about or at least about 60, 62, 64, 66, 68, 70, 72, 74, or75° C. In some embodiments, an antibody or antigen-binding fragmentthereof has a T_(m) of about 60° C. or greater.

In some embodiments, an antibody or antigen-binding fragment thereofconjugated to one or more cytotoxic or chemotherapeutic agents. Generalexamples of cytotoxic or chemotherapeutic agents include, withoutlimitation, alkylating agents, anti-metabolites, anthracyclines,anti-tumor antibiotics, platinums, type I topoisomerase inhibitors, typeII topoisomerase inhibitors, vinca alkaloids, and taxanes. Specificexamples of cytotoxic or chemotherapeutic agents include, withoutlimitation, cyclophosphamide, cilengitide, lomustine (CCNU), melphalan,procarbazine, carmustine (BCNU), enzastaurin, busulfan, daunorubicin,doxorubicin, gefitinib, erlotinib idarubicin, temozolomide, epirubicin,mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin,camptothecins, irinotecan, topotecan, amsacrine, etoposide, etoposidephosphate, teniposide, temsirolimus, everolimus, vincristine,vinblastine, vinorelbine, vindesine, CT52923, paclitaxel, imatinib,dasatinib, sorafenib, pazopanib, sunitnib, vatalanib, geftinib,erlotinib, AEE-788, dichoroacetate, tamoxifen, fasudil, SB-681323,semaxanib, donepizil, galantamine, memantine, rivastigmine, tacrine,rasigiline, naltrexone, lubiprostone, safinamide, istradefylline,pimavanserin, pitolisant, isradipine, pridopidine (ACR16),tetrabenazine, bexarotene, glatirimer acetate, fingolimod, andmitoxantrone, including pharmaceutically acceptable salts and acidsthereof. Further examples of cytotoxic or chemotherapeutic agentsinclude alkylating agents such as thiotepa, cyclophosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®., Rhne-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

The antibodies or antigen-binding fragments thereof can be used in anyof the compositions, methods, and/or kits described herein, and combinedwith one or more of the immunotherapy agents described herein.

Additional Therapeutic Agents and Compositions

Immunotherapy Agents.

Certain embodiments employ one or more cancer immunotherapy agents. Incertain instances, an immunotherapy agent modulates the immune responseof a subject, for example, to increase or maintain a cancer-related orcancer-specific immune response, and thereby results in increased immunecell inhibition or reduction of cancer cells. Exemplary immunotherapyagents include polypeptides, for example, antibodies and antigen-bindingfragments thereof, ligands, and small peptides, and mixtures thereof.Also include as immunotherapy agents are small molecules, cells (e.g.,immune cells such as T-cells), various cancer vaccines, gene therapy orother polynucleotide-based agents, including viral agents such asoncolytic viruses, and others known in the art. Thus, in certainembodiments, the cancer immunotherapy agent is selected from one or moreof immune checkpoint modulatory agents, cancer vaccines, oncolyticviruses, cytokines, and a cell-based immunotherapies.

In certain embodiments, the cancer immunotherapy agent is an immunecheckpoint modulatory agent. Particular examples include “antagonists”of one or more inhibitory immune checkpoint molecules, and “agonists” ofone or more stimulatory immune checkpoint molecules. Generally, immunecheckpoint molecules are components of the immune system that eitherturn up a signal (co-stimulatory molecules) or turn down a signal, thetargeting of which has therapeutic potential in cancer because cancercells can perturb the natural function of immune checkpoint molecules(see, e.g., Sharma and Allison, Science. 348:56-61, 2015; Topalian etal., Cancer Cell. 27:450-461, 2015; Pardoll, Nature Reviews Cancer.12:252-264, 2012). In some embodiments, the immune checkpoint modulatoryagent (e.g., antagonist, agonist) “binds” or “specifically binds” to theone or more immune checkpoint molecules, as described herein.

In particular embodiments, the immune checkpoint modulatory agent is apolypeptide or peptide. The terms “peptide” and “polypeptide” are usedinterchangeably herein, however, in certain instances, the term“peptide” can refer to shorter polypeptides, for example, polypeptidesthat consist of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, including allintegers and ranges (e.g., 5-10, 8-12, 10-15) in between. Polypeptidesand peptides can be composed of naturally-occurring amino acids and/ornon-naturally occurring amino acids, as described herein

Antibodies are also included as polypeptides. Thus, in some embodiments,the immune checkpoint modulatory polypeptide agent is an antibody or“antigen-binding fragment thereof”, as described elsewhere herein.

In some embodiments, the agent is or comprises a “ligand,” for example,a natural ligand, of the immune checkpoint molecule. A “ligand” refersgenerally to a substance or molecule that forms a complex with a targetmolecule (e.g., biomolecule) to serve a biological purpose, and includesa “protein ligand,” which generally produces a signal by binding to asite on a target molecule or target protein. Thus, certain agents areprotein ligands that, in nature, bind to an immune checkpoint moleculeand produce a signal. Also included are “modified ligands,” for example,protein ligands that are fused to a pharmacokinetic modifier, forexample, an Fc region derived from an immunoglobulin.

The binding properties of polypeptides can be quantified using methodswell known in the art (see Davies et al., Annual Rev. Biochem.59:439-473, 1990). In some embodiments, a polypeptide specifically bindsto a target molecule, for example, an immune checkpoint molecule or anepitope thereof, with an equilibrium dissociation constant that is aboutor ranges from about 1.0-7 to about 10-8 M. In some embodiments, theequilibrium dissociation constant is about or ranges from about 1.0-9 Mto about 1.0-10 M. In certain illustrative embodiments, the polypeptidehas an affinity (Kd or EC₅₀) for a target described herein (to which itspecifically binds) of about, at least about, or less than about, 0.01,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, or 50 nM.

In some embodiments, the agent is a “small molecule,” which refers to anorganic compound that is of synthetic or biological origin(biomolecule), but is typically not a polymer. Organic compounds referto a large class of chemical compounds whose molecules contain carbon,typically excluding those that contain only carbonates, simple oxides ofcarbon, or cyanides. A “biomolecule” refers generally to an organicmolecule that is produced by a living organism, including largepolymeric molecules (biopolymers) such as peptides, polysaccharides, andnucleic acids as well, and small molecules such as primary secondarymetabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids,vitamins, and hormones. A “polymer” refers generally to a large moleculeor macromolecule composed of repeating structural units, which aretypically connected by covalent chemical bond.

In certain embodiments, a small molecule has a molecular weight of aboutor less than about 1000-2000 Daltons, typically between about 300 and700 Daltons, and including about or less than about 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800,950, 1000 or 2000 Daltons.

Certain small molecules can have the “specific binding” characteristicsdescribed for herein polypeptides such as antibodies. For instance, insome embodiments a small molecule specifically binds to a target, forexample, an immune checkpoint molecule, with a binding affinity (Kd orEC₅₀) of about, at least about, or less than about, 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, or 50 nM.

In some embodiments, the immune checkpoint modulatory agent is anantagonist or inhibitor of one or more inhibitory immune checkpointmolecules. Exemplary inhibitory immune checkpoint molecules includeProgrammed Death-Ligand 1 (PD-L1), Programmed Death-Ligand 2 (PD-L2),Programmed Death 1 (PD-1), Cytotoxic T-Lymphocyte-Associated protein 4(CTLA-4), Indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase(TDO), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3),Lymphocyte Activation Gene-3 (LAG-3), V-domain Ig suppressor of T cellactivation (VISTA), B and T Lymphocyte Attenuator (BTLA), CD160, andT-cell immunoreceptor with Ig and ITIM domains (TIGIT).

In certain embodiments, the agent is a PD-1 (receptor) antagonist orinhibitor, the targeting of which has been shown to restore immunefunction in the tumor environment (see, e.g., Phillips et al., IntImmunol. 27:39-46, 2015). PD-1 is a cell surface receptor that belongsto the immunoglobulin superfamily and is expressed on T cells and pro-Bcells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functionsas an inhibitory immune checkpoint molecule, for example, by reducing orpreventing the activation of T-cells, which in turn reduces autoimmunityand promotes self-tolerance. The inhibitory effect of PD-1 isaccomplished at least in part through a dual mechanism of promotingapoptosis in antigen specific T-cells in lymph nodes while also reducingapoptosis in regulatory T cells (suppressor T cells). Some examples ofPD-1 antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to PD-1 and reducesone or more of its immune-suppressive activities, for example, itsdownstream signaling or its interaction with PD-L1. Specific examples ofPD-1 antagonists or inhibitors include the antibodies nivolumab,pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab, andantigen-binding fragments thereof (see, e.g., U.S. Pat. Nos. 8,008,449;8,993,731; 9,073,994; 9,084,776; 9,102,727; 9,102,728; 9,181,342;9,217,034; 9,387,247; 9,492,539; 9,492,540; and U.S. Application Nos.2012/0039906; 2015/0203579).

In some embodiments, the agent is a PD-L1 antagonist or inhibitor. Asnoted above, PD-L1 is one of the natural ligands for the PD-1 receptor.General examples of PD-L1 antagonists or inhibitors include an antibodyor antigen-binding fragment or small molecule that specifically binds toPD-L1 and reduces one or more of its immune-suppressive activities, forexample, its binding to the PD-1 receptor. Specific examples of PD-L1antagonists include the antibodies atezolizumab (MPDL3280A), avelumab(MSB0010718C), and durvalumab (MEDI4736), and antigen-binding fragmentsthereof (see, e.g., U.S. Pat. Nos. 9,102,725; 9,393,301; 9,402,899;9,439,962).

In some embodiments, the agent is a PD-L2 antagonist or inhibitor. Asnoted above, PD-L2 is one of the natural ligands for the PD-1 receptor.General examples of PD-L2 antagonists or inhibitors include an antibodyor antigen-binding fragment or small molecule that specifically binds toPD-L2 and reduces one or more of its immune-suppressive activities, forexample, its binding to the PD-1 receptor.

In some embodiments, the agent is a CTLA-4 antagonist or inhibitor.CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), alsoknown as CD152 (cluster of differentiation 152), is a protein receptorthat functions as an inhibitory immune checkpoint molecule, for example,by transmitting inhibitory signals to T-cells when it is bound to CD80or CD86 on the surface of antigen-presenting cells. General examplesCTLA-4 antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to CTLA-4. Particularexamples include the antibodies ipilimumab and tremelimumab, andantigen-binding fragments thereof. At least some of the activity ofipilimumab is believed to be mediated by antibody-dependentcell-mediated cytotoxicity (ADCC) killing of suppressor Tregs thatexpress CTLA-4.

In some embodiments, the agent is an IDO antagonist or inhibitor, or aTDO antagonist or inhibitor. IDO and TDO are tryptophan catabolicenzymes with immune-inhibitory properties. For example, IDO is known tosuppress T-cells and NK cells, generate and activate Tregs andmyeloid-derived suppressor cells, and promote tumor angiogenesis.General examples of IDO and TDO antagonists or inhibitors include anantibody or antigen-binding fragment or small molecule that specificallybinds to IDO or TDO (see, e.g., Platten et al., Front Immunol. 5: 673,2014) and reduces or inhibits one or more immune-suppressive activities.Specific examples of IDO antagonists or inhibitors include indoximod(NLG-8189), 1-methyl-tryptophan (1MT), β-Carboline (norharmane;9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g.,Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples ofTDO antagonists or inhibitors include 680C91 and LM10 (see, e.g.,Pilotte et al., PNAS USA. 109:2497-2502, 2012).

In some embodiments, the agent is a TIM-3 antagonist or inhibitor.T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) is expressed onactivated human CD4+ T-cells and regulates Th1 and Th17 cytokines. TIM-3also acts as a negative regulator of Th1/Tc1 function by triggering celldeath upon interaction with its ligand, galectin-9. TIM-3 contributes tothe suppressive tumor microenvironment and its overexpression isassociated with poor prognosis in a variety of cancers (see, e.g., Li etal., Acta Oncol. 54:1706-13, 2015). General examples of TIM-3antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to TIM-3 and reducesor inhibits one or more of its immune-suppressive activities.

In some embodiments, the agent is a LAG-3 antagonist or inhibitor.Lymphocyte Activation Gene-3 (LAG-3) is expressed on activated T-cells,natural killer cells, B-cells and plasmacytoid dendritic cells. Itnegatively regulates cellular proliferation, activation, and homeostasisof T-cells, in a similar fashion to CTLA-4 and PD-1 (see, e.g., Workmanand Vignali. European Journal of Immun. 33: 970-9, 2003; and Workman etal., Journal of Immun. 172: 5450-5, 2004), and has been reported to playa role in Treg suppressive function (see, e.g., Huang et al., Immunity.21: 503-13, 2004). LAG3 also maintains CD8+ T-cells in a tolerogenicstate and combines with PD-1 to maintain CD8 T-cell exhaustion. Generalexamples of LAG-3 antagonists or inhibitors include an antibody orantigen-binding fragment or small molecule that specifically binds toLAG-3 and inhibits one or more of its immune-suppressive activities.Specific examples include the antibody BMS-986016, and antigen-bindingfragments thereof.

In some embodiments, the agent is a VISTA antagonist or inhibitor.V-domain Ig suppressor of T cell activation (VISTA) is primarilyexpressed on hematopoietic cells and is an inhibitory immune checkpointregulator that suppresses T-cell activation, induces Foxp3 expression,and is highly expressed within the tumor microenvironment where itsuppresses anti-tumor T cell responses (see, e.g., Lines et al., CancerRes. 74:1924-32, 2014). General examples of VISTA antagonists orinhibitors include an antibody or antigen-binding fragment or smallmolecule that specifically binds to VISTA and reduces one or more of itsimmune-suppressive activities.

In some embodiments, the agent is a BTLA antagonist or inhibitor. B- andT-lymphocyte attenuator (BTLA; CD272) expression is induced duringactivation of T-cells, and it inhibits T-cells via interaction withtumor necrosis family receptors (TNF-R) and B7 family of cell surfacereceptors. BTLA is a ligand for tumor necrosis factor (receptor)superfamily, member 14 (TNFRSF14), also known as herpes virus entrymediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immuneresponses, for example, by inhibiting the function of human CD8+cancer-specific T-cells (see, e.g., Derré et al., J Clin Invest120:157-67, 2009). General examples of BTLA antagonists or inhibitorsinclude an antibody or antigen-binding fragment or small molecule thatspecifically binds to BTLA-4 and reduce one or more of itsimmune-suppressive activities.

In some embodiments, the agent is an HVEM antagonist or inhibitor, forexample, an antagonist or inhibitor that specifically binds to HVEM andinterferes with its interaction with BTLA or CD160. General examples ofHVEM antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to HVEM, optionallyreduces the HVEM/BTLA and/or HVEM/CD160 interaction, and thereby reducesone or more of the immune-suppressive activities of HVEM.

In some embodiments, the agent is a CD160 antagonist or inhibitor, forexample, an antagonist or inhibitor that specifically binds to CD160 andinterferes with its interaction with HVEM. General examples of CD160antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to CD160, optionallyreduces the CD160/HVEM interaction, and thereby reduces or inhibits oneor more of its immune-suppressive activities.

In some embodiments, the agent is a TIGIT antagonist or inhibitor. Tcell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor that isfound on the surface of a variety of lymphoid cells, and suppressesantitumor immunity, for example, via Tregs (Kurtulus et al., J ClinInvest. 125:4053-4062, 2015). General examples of TIGIT antagonists orinhibitors include an antibody or antigen-binding fragment or smallmolecule that specifically binds to TIGIT and reduce one or more of itsimmune-suppressive activities (see, e.g., Johnston et al., Cancer Cell.26:923-37, 2014).

In certain embodiments, the immune checkpoint modulatory agent is anagonist of one or more stimulatory immune checkpoint molecules.Exemplary stimulatory immune checkpoint molecules include OX40, CD40,Glucocorticoid-Induced TNFR Family Related Gene (GITR), CD137 (4-1BB),CD27, CD28, CD226, and Herpes Virus Entry Mediator (HVEM).

In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotesthe expansion of effector and memory T cells, and suppresses thedifferentiation and activity of T-regulatory cells (see, e.g., Croft etal., Immunol Rev. 229:173-91, 2009). Its ligand is OX4OL (CD252). SinceOX40 signaling influences both T-cell activation and survival, it playsa key role in the initiation of an anti-tumor immune response in thelymph node and in the maintenance of the anti-tumor immune response inthe tumor microenvironment. General examples of OX40 agonists include anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds to OX40 and increases one or more of itsimmunostimulatory activities. Specific examples include OX86, OX-40L,Fc-OX4OL, GSK3174998, MEDI0562 (a humanized OX40 agonist), MEDI6469(murine OX4 agonist), and MEDI6383 (an OX40 agonist), andantigen-binding fragments thereof.

In some embodiments, the agent is a CD40 agonist. CD40 is expressed onantigen-presenting cells (APC) and some malignancies. Its ligand isCD40L (CD154). On APC, ligation results in upregulation of costimulatorymolecules, potentially bypassing the need for T-cell assistance in anantitumor immune response. CD40 agonist therapy plays an important rolein APC maturation and their migration from the tumor to the lymph nodes,resulting in elevated antigen presentation and T cell activation.Anti-CD40 agonist antibodies produce substantial responses and durableanticancer immunity in animal models, an effect mediated at least inpart by cytotoxic T-cells (see, e.g., Johnson et al. Clin Cancer Res.21: 1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res.19:1035-43, 2013). General examples of CD40 agonists include an antibodyor antigen-binding fragment or small molecule or ligand thatspecifically binds to CD40 and increases one or more of itsimmunostimulatory activities. Specific examples include CP-870,893,dacetuzumab, Chi Lob 7/4, ADC-1013, CD40L, rhCD40L, and antigen-bindingfragments thereof.

In some embodiments, the agent is a GITR agonist. Glucocorticoid-InducedTNFR family Related gene (GITR) increases T cell expansion, inhibits thesuppressive activity of Tregs, and extends the survival of T-effectorcells. GITR agonists have been shown to promote an anti-tumor responsethrough loss of Treg lineage stability (see, e.g., Schaer et al., CancerImmunol Res. 1:320-31, 2013). These diverse mechanisms show that GITRplays an important role in initiating the immune response in the lymphnodes and in maintaining the immune response in the tumor tissue. Itsligand is GITRL. General examples of GITR agonists include an antibodyor antigen-binding fragment or small molecule or ligand thatspecifically binds to GITR and increases one or more of itsimmunostimulatory activities. Specific examples include GITRL,INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is amember of the tumor necrosis factor (TNF) receptor family, andcrosslinking of CD137 enhances T-cell proliferation, IL-2 secretion,survival, and cytolytic activity. CD137-mediated signaling also protectsT-cells such as CD8+ T-cells from activation-induced cell death. Generalexamples of CD137 agonists include an antibody or antigen-bindingfragment or small molecule or ligand that specifically binds to CD137and increases one or more of its immunostimulatory activities. Specificexamples include the CD137 (or 4-1BB) ligand (see, e.g., Shao andSchwarz, J Leukoc Biol. 89:21-9, 2011) and the antibody utomilumab,including antigen-binding fragments thereof.

In some embodiments, the agent is a CD27 agonist. Stimulation of CD27increases antigen-specific expansion of naive T cells and contributes toT-cell memory and long-term maintenance of T-cell immunity. Its ligandis CD70. The targeting of human CD27 with an agonist antibody stimulatesT-cell activation and antitumor immunity (see, e.g., Thomas et al.,Oncoimmunology. 2014; 3:e27255. doi:10.4161/onci.27255; and He et al., JImmunol. 191:4174-83, 2013). General examples of CD27 agonists includean antibody or antigen-binding fragment or small molecule or ligand thatspecifically binds to CD27 and increases one or more of itsimmunostimulatory activities. Specific examples include CD70 and theantibodies varlilumab and CDX-1127 (1F5), including antigen-bindingfragments thereof.

In some embodiments, the agent is a CD28 agonist. CD28 is constitutivelyexpressed CD4+ T cells some CD8+ T cells. Its ligands include CD80 andCD86, and its stimulation increases T-cell expansion. General examplesof CD28 agonists include an antibody or antigen-binding fragment orsmall molecule or ligand that specifically binds to CD28 and increasesone or more of its immunostimulatory activities. Specific examplesinclude CD80, CD86, the antibody TAB08, and antigen-binding fragmentsthereof.

In some embodiments, the agent is CD226 agonist. CD226 is a stimulatingreceptor that shares ligands with TIGIT, and opposite to TIGIT,engagement of CD226 enhances T-cell activation (see, e.g., Kurtulus etal., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med.1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538,2004). General examples of CD226 agonists include an antibody orantigen-binding fragment or small molecule or ligand (e.g., CD112,CD155) that specifically binds to CD226 and increases one or more of itsimmunostimulatory activities.

In some embodiments, the agent is an HVEM agonist. Herpesvirus entrymediator (HVEM), also known as tumor necrosis factor receptorsuperfamily member 14 (TNFRSF14), is a human cell surface receptor ofthe TNF-receptor superfamily. HVEM is found on a variety of cellsincluding T-cells, APCs, and other immune cells. Unlike other receptors,HVEM is expressed at high levels on resting T-cells and down-regulatedupon activation. It has been shown that HVEM signaling plays a crucialrole in the early phases of T-cell activation and during the expansionof tumor-specific lymphocyte populations in the lymph nodes. Generalexamples of HVEM agonists include an antibody or antigen-bindingfragment or small molecule or ligand that specifically binds to HVEM andincreases one or more of its immunostimulatory activities.

In certain embodiments, the cancer immunotherapy agent is a cancervaccine. Exemplary cancer vaccines include Oncophage, humanpapillomavirus HPV vaccines such Gardasil or Cervarix, hepatitis Bvaccines such as Engerix-B, Recombivax HB, or Twinrix, and sipuleucel-T(Provenge). In some embodiments, the cancer vaccine comprises orutilizes one or more cancer antigens, or cancer-associate d antigens.Exemplary cancer antigens include, without limitation, human Her2/neu,HerVEGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22,CD23 (IgE Receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascularendothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, VEGR-3,NRP2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152,CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin,insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein,insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX),carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53,survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembraneglycoprotein NMB, fibroblast activation protein alpha (FAP),glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine,prostate-specific membrane antigen (PSMA), NR-LU-13 antigen, TRAIL-R1,tumor necrosis factor receptor superfamily member 10b (TNFRSF10B orTRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pancarcinoma antigen,B-cell activating factor (BAFF), platelet-derived growth factorreceptor, glycoprotein EpCAM (17-1A), Programmed Death-1, proteindisulfide isomerase (PDI), Phosphatase of Regenerating Liver 3 (PRL-3),prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialogangliosideexpressed on tumors of neuroectodermal origin), glypican-3 (GPC3), andmesothelin.

In certain embodiments, the cancer immunotherapy agent is an oncolyticvirus. An oncolytic virus is a virus that preferentially infects andkills cancer cells. Included are naturally-occurring and man-made orengineered oncolytic viruses. Most oncolytic viruses are engineered fortumor selectivity, although there are naturally-occurring examples suchas Reovirus and the SVV-001 Seneca Valley virus. General examples ofoncolytic viruses include VSV, Poliovirus, Reovirus, Senecavirus, andRIGVIR, and engineered versions thereof. Non-limiting examples ofoncolytic viruses include herpes simplex virus (HSV) and engineeredversion thereof, talimogene laherparepvec (T-VEC), coxsackievirus A21(CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus(NTX-010), Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102(Ad5/3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401, among others.

In certain embodiments, the cancer immunotherapy agent is a cytokine.Exemplary cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7,IL-21, and Granulocyte-macrophage colony-stimulating factor (GM-CSF).

In certain embodiments, the cancer immunotherapy agent is cell-basedimmunotherapy, for example, a T-cell based adoptive immunotherapy. Insome embodiments, the cell-based immunotherapy comprises cancerantigen-specific T-cells, optionally ex vivo-derived T-cells. In someembodiments, the cancer antigen-specific T-cells are selected from oneor more of chimeric antigen receptor (CAR)-modified T-cells, and T-cellReceptor (TCR)-modified T-cells, tumor infiltrating lymphocytes (TILs),and peptide-induced T-cells. In specific embodiments, the CAR-modifiedT-cell is targeted against CD-19 (see, e.g., Maude et al., Blood.125:4017-4023, 2015).

In certain instances, the cancer to be treated associates with thecancer antigen, that is, the cancer antigen-specific T-cells aretargeted against or enriched for at least one antigen that is known toassociate with the cancer to be treated. In some embodiments, the cancerantigen is selected from one or more of CD19, human Her2/neu, HerVEGFreceptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD20, CD22, CD23 (IgEReceptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelialgrowth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37,CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4,HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1(IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA),guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrinα5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblastactivation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (orCA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA),NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamilymember 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin.

Additional exemplary cancer antigens include 5T4, 707-AP, 9D7, AFP,AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin,alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m,B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m,CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m,cathepsin B, cathepsin L, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28,CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6,Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN,EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1,EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2,hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R1 71, HLA-A11/m, HLA-A2/m,HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7,HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immaturelaminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205,KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1,MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B1 6,MAGE-B1 7, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4,MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A,MART-1/melan-A, MART-2, MART-2/m, matrix protein 22, MCI R, M-CSF,ME1/m, mesothelin, MG50/PXDN, MMP1 1, MN/CA IX-antigen, MRP-3, MUC-1,MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A,N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP,NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, NY-ESO-1, OA1, OFA-iLRP, OGT,OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, pi 5, p190 minor bcr-abl,p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 Kinase,Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3, PSA,PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD1 68, RU1,RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp1 7, SSX-1,SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B,SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF-beta,TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2,TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, and WT1. Certainpreferred antigens include p53, CA125, EGFR, Her2/neu, hTERT, PAP,MAGE-A1, MAGE-A3, Mesothelin, MUC-1, GP100, MART-1, Tyrosinase, PSA,PSCA, PSMA, STEAP-1, Ras, CEA and WT1, and more preferably PAP, MAGE-A3,WT1, and MUC-1.

In some embodiments the antigen is selected from MAGE-A1 (e.g., MAGE-A1according to accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g.,MAGE-A6 according to accession number NM_005363), MAGE-C1, MAGE-C2,melan-A (e.g., melan-A according to accession number NM_00551 1), GP100(e.g., GP100 according to accession number M77348), tyrosinase (e.g.,tyrosinase according to accession number NM_000372), survivin (e.g.,survivin according to accession number AF077350), CEA (e.g., CEAaccording to accession number NM_004363), Her-2/neu (e.g., Her-2/neuaccording to accession number M1 1 730), WT1 (e.g., WT1 according toaccession number NM_000378), PRAME (e.g., PRAME according to accessionnumber NM_0061 15), EGFRI (epidermal growth factor receptor 1) (e.g.,EGFRI (epidermal growth factor receptor 1) according to accession numberAF288738), MUC1, mucin-1 (e.g., mucin-1 according to accession numberNM_002456), SEC61 G (e.g., SEC61 G according to accession numberNM_014302), hTERT (e.g., hTERT accession number NM_198253), 5T4 (e.g.,5T4 according to accession number NM_006670), TRP-2 (e.g., TRP-2according to accession number NM_001 922), STEAP1 (Six-transmembraneepithelial antigen of prostate 1), PSCA, PSA, PSMA, etc.

In some embodiments, the cancer antigen is selected from PCA, PSA, PSMA,STEAP, and optionally MUC-1, including fragments, variants, andderivatives thereof. In some embodiments, the cancer antigen selectedfrom NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and optionally MUC-1,including fragments, variants, and derivatives thereof.

In some instances, cancer antigens encompass idiotypic antigensassociated with a cancer or tumor disease, particularly lymphoma or alymphoma associated disease, for example, wherein the idiotypic antigenis an immunoglobulin idiotype of a lymphoid blood cell or a T cellreceptor idiotype of a lymphoid blood cell.

In some instances, the cancer antigen-specific T-cells are selected fromone or more of chimeric antigen receptor (CAR)-modified T-cells (e.g.,targeted against a cancer antigen), and T-cell Receptor (TCR)-modifiedT-cells, tumor infiltrating lymphocytes (TILs), and peptide-inducedT-cells.

The skilled artisan will appreciate that the various cancerimmunotherapy agents described herein can be combined with any one ormore of the various anti-NRP2 antibodies (including antigen-bindingfragments thereof) described herein, and used according to any one ormore of the methods or compositions described herein.

Chemotherapeutic Agents.

Certain embodiments employ one or more chemotherapeutic agents, forexample, small molecule chemotherapeutic agents. Non-limiting examplesof chemotherapeutic agents include alkylating agents, anti-metabolites,cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), ananti-microtubule agents, among others.

Examples of alkylating agents include nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil,ifosfamide, and busulfan), nitrosoureas (e.g., N-Nitroso-N-methylurea(MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU),fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine,mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mytomycin,and diaziquone (AZQ)), cisplatins and derivatives thereof (e.g.,carboplatin and oxaliplatin), and non-classical alkylating agents(optionally procarbazine and hexamethylmelamine).

Examples of anti-metabolites include anti-folates (e.g., methotrexateand pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil andcapecitabine), deoxynucleoside analogues (e.g., ancitabine, enocitabine,cytarabine, gemcitabine, decitabine, azacitidine, fludarabine,nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), andthiopurines (e.g., thioguanine and mercaptopurine);

Examples of cytotoxic antibiotics include anthracyclines (e.g.,doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin,aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone,and actinomycin. Examples of topoisomerase inhibitors includecamptothecin, irinotecan, topotecan, etoposide, doxorubicin,mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.

Examples of anti-microtubule agents include taxanes (e.g., paclitaxeland docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine,vindesine, vinorelbine).

The skilled artisan will appreciate that the various chemotherapeuticagents described herein can be combined with any one or more of thevarious anti-NRP2 antibodies (including antigen-binding fragmentsthereof) described herein, and used according to any one or more of themethods or compositions described herein.

Hormonal Therapeutic Agents.

Certain embodiments employ at least one hormonal therapeutic agent.General examples of hormonal therapeutic agents include hormonalagonists and hormonal antagonists. Particular examples of hormonalagonists include progestogen (progestin), corticosteroids (e.g.,prednisolone, methylprednisolone, dexamethasone), insulin like growthfactors, VEGF derived angiogenic and lymphangiogenic factors (e.g.,VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growthfactor (FGF), galectin, hepatocyte growth factor (HGF), platelet derivedgrowth factor (PDGF), transforming growth factor (TGF)-beta, androgens,estrogens, and somatostatin analogs. Examples of hormonal antagonistsinclude hormone synthesis inhibitors such as aromatase inhibitors andgonadotropin-releasing hormone (GnRH)s agonists (e.g., leuprolide,goserelin, triptorelin, histrelin) including analogs thereof. Alsoincluded are hormone receptor antagonist such as selective estrogenreceptor modulators (SERMs; e.g., tamoxifen, raloxifene, toremifene) andanti-androgens (e.g., flutamide, bicalutamide, nilutamide).

Also included are hormonal pathway inhibitors such as antibodiesdirected against hormonal receptors. Examples include inhibitors of thethe IGF receptor (e.g., IGF-IR1) such as cixutumumab, dalotuzumab,figitumumab, ganitumab, istiratumab, and robatumumab; inhibitors of thevascular endothelial growth factor receptors 1, 2 or 3 (VEGFR1, VEGFR2or VEGFR3) such as alacizumab pegol, bevacizumab, icrucumab,ramucirumab; inhibitors of the TGF-beta receptors R1, R2, and R3 such asfresolimumab and metelimumab; inhibitors of c-Met such as naxitamab;inhibitors of the EGF receptor such as cetuximab, depatuxizumabmafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab,modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, andzalutumumab; inhibitors of the FGF receptor such as aprutumab ixadotinand bemarituzumab; and inhibitors of the PDGF receptor such asolaratumab and tovetumab.

The skilled artisan will appreciate that the various hormonaltherapeutic agents described herein can be combined with any one or moreof the various anti-NRP2 antibodies (including antigen-binding fragmentsthereof) described herein, and used according to any one or more of themethods or compositions described herein.

Kinase Inhibitors.

Certain embodiments employ at least one kinase inhibitor, includingtyrosine kinase inhibitors. Examples of kinase inhibitors include,without limitation, adavosertib, afanitib, aflibercept, axitinib,bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib,crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib,fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib,mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib,ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656,tofacitinib, trastuzumab, vandetanib, and vemuafenib.

The skilled artisan will appreciate that the various kinase inhibitorsdescribed herein can be combined with any one or more of the variousanti-NRP2 antibodies (including antigen-binding fragments thereof)described herein, and used according to any one or more of the methodsor compositions described herein.

Methods of Use and Therapeutic Compositions

Embodiments of the present disclosure relate to the discovery that humanhistidyl-tRNA synthetase (HRS) polypeptides have unexpected biologicalproperties which are relevant to treating a broad range of diseases andconditions, and that certain of these properties relate to theinteractions between HRS and human neuropilin 2 (NRP2). Accordingly,antibodies directed against human NRP2, which interfere with the bindingbetween NRP2 and NRP2 ligands, including, for example, human HRS, can beused as standalone therapies in the treatment of diseases, includingNRP2-associated diseases, or in combination with other therapeuticagents as described herein.

Certain embodiments therefore include methods of treating, amelioratingthe symptoms of, and/or reducing the progression of, a disease orcondition in a subject in need thereof, comprising administering to thesubject at least one antibody or antigen-binding fragment thereof thatspecifically binds to a human neuropilin-2 (NRP2) polypeptide. In someinstances, the at least one antibody or antigen-binding fragment thereofantagonizes the binding/signaling activity between the NRP2 polypeptideand the at least one NRP2 ligand. In some instances, the at least oneantibody or antigen-binding fragment thereof mimics or otherwiseenhances one or more signaling activities of the NRP2/NRP2 ligandinteraction, for example, by acting as an agonist antibody.

In some instances, the at least one antibody or antigen-binding fragmentthereof interferes with binding of the human NRP2 polypeptide to a humanHRS polypeptide. In some instances, the at least one antibody orantigen-binding fragment thereof mimics one or more signaling activitiesof the HRS polypeptide binding to the NRP2 polypeptide, for example, byacting as an agonist antibody. Exemplary anti-NRP2 antibodies andtherapeutic compositions comprising the same are described elsewhereherein.

In certain embodiments, the disease is or condition is anNRP2-associated disease or condition. In some embodiments, theNRP2-associated disease or condition is selected from one or more ofcancer and diseases and pathways associated with cancer, includingcancer cell growth, initiation, migration, adhesion, invasion, and/ormetastasis; diseases associated with inflammation, autoimmunity, andrelated inflammatory diseases, including diseases associated withinappropriate immune cell activation or migration such as Graft versushost disease (GVHD); diseases associated with lymphatic development,lymphangiogenesis, and lymphatic damage, including, for example, edema,lymphedema, secondary lymphedema, inappropriate fat absorption anddeposition, excess fat deposition, and vascular permeability; diseasesassociated with infections, including latent infections; diseasesassociated with allergic disorders/diseases, allergic responses,including, for example, chronic obstructive pulmonary disorder (COPD),neutrophilic asthma, antineutrophil cytoplasmic antibody(ANCA)-associated systemic vasculitis, systemic lupus erythematosus,rheumatoid arthritis, inflammasome-related diseases, and skin-relatedneutrophil-mediated diseases such as pyoderma gangrenosum; diseasesassociated with granulomatous inflammatory diseases, includingsarcoidosis and granulomas; diseases associated with fibrosis includingfibrotic diseases, fibrosis, endothelial to mesenchymal transition(EMT), and wound healing; diseases associated with inappropriate smoothmuscle contractility, and inappropriate vascular smooth muscle cellmigration and adhesion; diseases associated with inappropriateautophagy, phagocytosis, and efferocytosis; diseases associated withneuronal diseases, peripheral nervous system remodeling, and painperception; and diseases associated with bone development and boneremodeling.

In some embodiments, the disease is a cancer. Here, upregulation of NRP2expression is associated with tumorigenesis and in particular tumormetastasis, and is correlated with more aggressive disease in severaltumor types. Moreover, the semaphorin/plexin/neuropilin signaling axisinfluences many of the hallmarks of cancer (see, for example, Franzolinand Tamagnone Int. J. Mol. Sci. 20, 377; doi:10.3390/ijms20020377,2019); Nasarre et al., OncoTargets and Therapy 2014:7 1663-1687; Neufeldet al., Cold Spring Harb Perspect Med. 2:a006718, 2012). Consistent withthese studies, increased expression of NRP2 in prostate cancer cells isinduced by Phosphatase and tensin homolog (PTEN deletion), and itsexpression correlates with Gleason grade (see, for example, Zhao et al.,Thoracic Cancer 8: 203-213, 2017). Additionally, P53 mutationsupregulate NRP2 expression via suppression of DLX2 transcription leadingto increased cell mobility. About 50% of human tumors and cancerscontain a mutation in the p53 gene, with the vast majority of thesemutations occurring in the DNA binding domain, and such is linked topoorer prognosis (see, for example, Drabkin et al., Oncotarget. 8 (No57) 96464-96465, 2017). Additionally, TGF-β signaling is involved in theexpression of NRP2B, and up-regulation of EMT in cancer, which mayexplain why in advanced tumors, higher production of TGF-β is positivelyassociated with tumor aggressiveness and poor prognosis (see, forexample, Malfettone et al., Cancer Lett. 392:39-50, 2017).

Certain embodiments thus include methods of treating ameliorating thesymptoms of, or inhibiting the progression of, a cancer in a subject inneed thereof, comprising administering to the subject at least oneantibody or antigen-binding fragment thereof that specifically binds toa human NRP2 polypeptide (an anti-NRP2 antibody), and which modulates(e.g., interferes with) binding of the human NRP2 polypeptide to a NRP2ligand (for example, an NRP2 ligand from Table N2 or Table N3 and/or ahuman HRS polypeptide from Table H1). Certain embodiments includereducing or preventing the re-emergence of a cancer in a subject in needthereof, for example, a metastatic cancer, wherein administration of thetherapeutic composition enables generation of an immune memory to thecancer. In some embodiments, the subject has or is at risk fordeveloping diabetes, for example, type 1 diabetes or type 2 diabetes.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of at least one NRP2 ligand (for example, an NRP2 ligand fromTable N2 or Table N3 and/or an HRS polypeptide from Table H1) and/or acoding mRNA thereof relative to a healthy control. For instance, in someembodiments, the levels of the at least one NRP2 ligand in the diseasessubject, cells, or tissue are about or at least about 1.5, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or moretimes the levels of the at least one NRP2 ligand in a healthy control.In some embodiments, the subject has, and/or is selected for treatmentbased on having, a cancer which has increased levels or expression of atleast one NRP2 ligand (for example, an NRP2 ligand from Table N2 orTable N3 and/or an HRS polypeptide from Table H1) and/or a coding mRNAthereof relative to a non-cancerous control cell or tissue. Forinstance, in some embodiments, the levels of the at least one NRP2ligand in the cancer cells or tissue are about or at least about 1.5, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000or more times the levels of the NRP2 ligand in a non-cancerous controlor standard. Thus, certain embodiments include methods of selecting asubject for treatment, comprising (i) detecting increased expressionlevels of at least one NRP2 ligand (for example, an NRP2 ligand fromTable N2 or Table N3 and/or an HRS polypeptide from Table H1) and/orcoding mRNA in the subject relative to a control or reference, and (ii)administering to the subject a therapeutic composition comprising atleast one anti-NRP2-antibody, as described herein. In particularembodiments, the HRS polypeptide is a splice variant of full-length HRS.In some embodiments, the HRS splice variant is selected from one or moreof HisRS^(N1), HisRS^(N2), HisRS^(N3), HisRS^(N4), HisRS^(N5),HisRS^(C1), HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5), HisRS^(C6),HisRS^(C7), HisRS^(C8), and HisRS^(C9).

In some embodiments, the subject has, and/or is selected for treatmentbased on having, increased circulating or serum levels of a solubleneuropilin 2 (NRP2) polypeptide (selected, for example, from Table N1),either bound to an HRS polypeptide or free, relative to the levels of ahealthy or matched control population of subject(s). For instance, incertain embodiments, the circulating or serum levels are about or atleast about 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000,4000, 5000 pM of the soluble NRP2 polypeptide, or the circulating orserum levels are about 30-50, 50-100, 100-2000, 200-2000, 300-2000,400-2000, 500-2000, 600-2000, 700-2000, 800-2000, 900-2000, 1000-2000,2000-3000, 3000-4000, 4000-5000 pM of the soluble NRP2 polypeptide.

In certain embodiments, the subject has, and/or is selected fortreatment based on having, a disease associated with increased levels orexpression of an NRP2 polypeptide (optionally selected from Table N1)and/or a coding mRNA thereof relative to a healthy control (e.g., anNRP2-associated disease). For example, in certain embodiments, thelevels of the NRP2 polypeptide in the diseased subject, cells, or tissueare about or at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100 or more times the levels of NRP2 polypeptidein a healthy control. In some embodiments, the subject has, and/or isselected for treatment based on having, a cancer which has increasedlevels or expression of a NRP2 polypeptide (selected, for example, fromTable N1) and/or a coding mRNA thereof relative to a control cell ortissue, optionally relative to a non-cancerous cell or tissue of thesame type as the cancer. For instance, in some embodiments, the levelsof the NRP2 polypeptide in the cancer cells or tissue are about or atleast about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70,80, 90, 100 or more times the levels of NRP2 polypeptide in anon-cancerous control or standard. Some embodiments thus include methodsof selecting a subject for treatment, comprising (i) detecting increasedexpression levels of a NRP2 polypeptide and/or a coding mRNA thereof inthe subject relative to a control or reference, and (ii) administeringto the subject a therapeutic composition comprising at least oneanti-NRP2-antibody, as described herein.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of NRP2A and/or NRP2B, or an altered ratio of NRP2A:NRP2Bexpression, relative to a healthy control or matched control standard orpopulation of subject(s). In some embodiments, the subject hassignificantly higher expression or levels of NRP2B relative to a healthycontrol or matched control standard or population of subject(s). In someembodiments, the levels of NRP2B are increased by about or at leastabout 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%,800%, 900%, 1000% compared to a healthy control or matched controlstandard or population of subject(s). In some embodiments, the subjecthas, and/or is selected for treatment based on having, increasedcirculating levels of HRS:NRP2 complexes relative to a healthy ormatched control standard or population of subject(s). Certainembodiments therefore include methods of selecting a subject for cancertreatment, comprising (i) detecting increased expression levels ofHRS:NRP2 complexes in the subject relative to a control or reference,and (ii) administering to the subject a therapeutic compositioncomprising at least one anti-NRP2-antibody, as described herein.

In some embodiments, the healthy control or matched control standard orpopulation of subject(s) comprises average ranges for age-matchedsamples of cancerous or non-cancerous cells or tissue of the same typeas the cancer, which comprise specific characteristics such as drugresistance, metastatic potential, aggressiveness, genetic signature(e.g., p53 mutations, PTEN deletion, IGFR expression), and/or expressionpatterns.

Some embodiments comprise administering at least one anti-NRP2 antibodyto a subject in an amount and at a frequency sufficient to achieve anaverage, sustained blood plasma concentration of soluble NRP2 of aboutor less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pm, 40 pM,30 pM, 20 pM, or 10 pM.

Certain embodiments comprise administering at least one anti-NRP2antibody in an amount and at a frequency sufficient to achieve areduction in the circulating levels of HRS:NRP2 complexes, for example,a reduction of about or at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 60, 70, 80, 90, 95, 99, or 100%.

For the treatment of cancer, in some instances, an anti-NRP2 antibodyenhances the immune response to the cancer by about, or at least about,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000% or more, relative to anuntreated control. Exemplary immune responses include increasing orenhancing immune cell invasion of a solid tumor, and increasing thebiological activity against the cancer. In certain embodiments, ananti-NRP2 antibody enhances an adaptive immune response to the cancer,and in some embodiments, an anti-NRP2 antibody enhances an innate immuneresponse to the cancer. In some-instances, an anti-NRP2 antibodydirectly or indirectly enhances a T-cell-mediated response to thecancer. In some-instances, an anti-NRP2 antibody enhances aB-cell-mediated or antibody-mediated response to the cancer. Insome-instances, an anti-NRP2 antibody modulates a macrophage responsesto the cancer. In some-instances, an anti-NRP2 antibody modulates immunecell, or cancer autophagy. In some-instances, an anti-NRP2 antibodymodulates immune cell phagocytosis. In some-instances, an anti-NRP2antibody modulates cancer cell apoptosis. In some-instances, ananti-NRP2 antibody modulates immune cell efferocytosis and/or cancercell autophagy.

In some embodiments, an anti-NRP2 antibody enhances macrophage responsesto the cancer. In some embodiments, an anti-NRP2 inhibits macrophageresponses to the cancer. In some embodiments of the anti-NRP2 antibody,the antibody enhances autophagy. In some embodiments, an anti-NRP2inhibits autophagy. In some embodiments, an anti-NRP2 enhancesphagocytosis. In some embodiments, an anti-NRP2 inhibits phagocytosis.In some embodiments, an anti-NRP2 enhances apoptosis. In someembodiments of the anti-NRP2 antibody, the antibody inhibits apoptosis.In some embodiments, an anti-NRP2 antibody enhances efferocytosis. Insome embodiments, an anti-NRP2 inhibits efferocytosis.

In some-instances, an anti-NRP2 antibody reduces cancer initiation,cancer cell migration, adhesion, or cancer cell metastasis by about orat least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or morerelative to an untreated control. In some-instances, an anti-NRP2antibody reduces cancer mediated lymphoangiogenesis by about or at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to anuntreated control.

In some embodiments, the at least one anti-NRP2 antibody reduces therate of in vitro growth of the cancer (for example, cancer cellsisolated from a biopsy or other sample grown in vitro) by about or atleast about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative toan untreated control.

In some embodiments, the at least one anti-NRP2 antibody reduces theadhesiveness of the cancer (for example, cancer cells isolated from abiopsy or other sample grown in vitro) to a substrate by about or atleast about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative toan untreated control. In some instances, the substrate compriseslaminin.

In some embodiments, the at least one anti-NRP2 antibody reduces theinvasiveness of the cancer (for example, cancer cells isolated from abiopsy or other sample grown in vitro) by about or at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000% or more relative to an untreatedcontrol.

In some embodiments, the at least one anti-NRP2 antibody inhibits therate of migration or motility of the cancer or a migratory cell (forexample, cancer or immune cells isolated from a biopsy or other samplegrown in vitro) by about or at least about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000% or more relative to an untreated control.

In some embodiments, the at least one anti-NRP2 antibody inhibits therate of autophagy or endosome maturation (for example, endosomeacidification) of the cancer or associated immune cells by about or atleast about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative toan untreated control.

In some embodiments, the at least one anti-NRP2 antibody enhances thesusceptibility of the cancer to an additional agent (for example,chemotherapeutic agent, hormonal therapeutic agent, and or kinaseinhibitor) by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000% or more relative to the additional agent alone. In someembodiments, the at least one anti-NRP2 antibody enhances an anti-tumorand/or immunostimulatory activity of a cancer immunotherapy agent byabout or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% ormore, relative to the cancer immunotherapy agent alone.

Some embodiments include administering the at least one anti-NRP2antibody in an amount and at a frequency sufficient to achieve a steadystate concentration, or average circulating concentration, of the atleast one anti-NRP2 antibody of between about 1 nM and about 1 μM,between about 1 nM and about 100 nM, between about 1 nM and about 10 nM,or between about 1 nM and about 3 μM.

Also include are combination therapies for treating cancers, includingmethods of treating ameliorating the symptoms of, or inhibiting theprogression of, a cancer in a subject in need thereof, comprisingadministering to the subject at least one antibody or antigen-bindingfragment thereof that specifically binds to a human NRP2 polypeptide (ananti-NRP2 antibody) in combination with at least one additional agent,for example, a cancer immunotherapy agent, a chemotherapeutic agent, ahormonal therapeutic agent, and/or a kinase inhibitor. Exemplary cancerimmunotherapy agents, chemotherapeutic agents, hormonal therapeuticagents, and kinase inhibitors are described elsewhere herein.

In some instances, an anti-NRP2 antibody and the at least one additionalagent are administered separately, for example, in separate therapeuticcompositions and at the same or different times. In some embodiments, ananti-NRP2 antibody and the at least one additional agent areadministered as part of the same therapeutic composition, at the sametime.

Particular methods employ one or more anti-NRP2 antibodies, orantigen-binding fragments thereof, as part of (i.e., in addition to) acombination therapy regimen. Exemplary combination regiments areprovided in Table M1 below.

TABLE M1 Combination Therapy Regimens Cancer Type Agents Acronym BreastCyclophosphamide, methotrexate, CMF Cancer 5-fluorouracil, vinorelbineDoxorubicin, cyclophosphamide AC Hodgkin's Docetaxel, doxorubicin, TAClymphoma cyclophosphamide Doxorubicin, bleomycin, ABVD vinblastine,dacarbazine Mustine, vincristine, MOPP procarbazine, prednisolone Non-Cyclophosphamide, doxorubicin, CHOP Hodgkin's vincristine, prednisolonelymphoma Germ cell Bleomycin, etoposide, cisplatin BEP tumor StomachEpirubicin, cisplatin, 5-fluorouracil ECF cancer Epirubicin, cisplatin,capecitabine ECX Bladder Methotrexate, vincristine, MVAC cancerdoxorubicin, cisplatin Lung Cyclophosphamide, doxorubicin, CAV cancervincristine, vinorelbine Colorectal 5-fluorouracil, folinic acid, FOLFOXcancer oxaliplatin Pancreatic Leucovorin, fluorouracil, irinotecanFOLFIRINOX Cancer (Camptosar), oxaliplatin Gemcitabine, nabpaclitaxelABRAXANE

In some embodiments, the methods and therapeutic compositions describedherein (for example, anti-NRP2 antibody, alone or in combination with atleast one additional agent) increase median survival time of a subjectby 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certainembodiments, the methods and therapeutic compositions described herein(for example, anti-NRP2 antibody, alone or in combination with at leastone additional agent) increase median survival time of a subject by 1year, 2 years, 3 years, or longer. In some embodiments, the methods andtherapeutic compositions described herein (for example, anti-NRP2antibody, alone or in combination with cancer immunotherapy agent)increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. Incertain embodiments, the methods or therapeutic compositions describedherein increase progression-free survival by 1 year, 2 years, 3 years,or longer.

In certain embodiments, the methods and therapeutic compositionsdescribed herein (for example, anti-NRP2 antibody, alone or incombination with at least one additional agent) are sufficient to resultin tumor regression, as indicated by a statistically significantdecrease in the amount of viable tumor, for example, at least a 10%,20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered(e.g., decreased with statistical significance) scan dimensions. Incertain embodiments, the methods and therapeutic compositions describedherein (for example, anti-NRP2 antibody, alone or in combination with atleast one additional agent) are sufficient to result in stable disease.In certain embodiments, the methods and therapeutic compositionsdescribed herein (for example, anti-NRP2 antibody, alone or incombination with cancer immunotherapy agent) are sufficient to result inclinically relevant reduction in symptoms of a particular diseaseindication known to the skilled clinician.

In some embodiments, an anti-NRP2 antibody increases, complements, orotherwise enhances the anti-tumor and/or immunostimulatory activity ofthe cancer immunotherapy agent, relative to the cancer immunotherapyagent alone. In some embodiments, an anti-NRP2 antibody enhances theanti-tumor and/or immunostimulatory activity of the cancer immunotherapyagent by about, or at least about, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000% or more, relative to the cancer immunotherapy agent alone.

The methods and therapeutic compositions described herein can be used inthe treatment of any variety of cancers or tumors. In some embodiments,the cancer is a primary cancer, i.e., a cancer growing at the anatomicalsite where tumor progression began and yielded a cancerous mass. In someembodiments, the cancer is a secondary or metastatic cancer, i.e., acancer which has spread from the primary site or tissue of origin intoone or more different sites or tissues. In some embodiments, the cancerexpresses or overexpresses NRP2. In some embodiments, the subject orpatient has a cancer selected from one or more of melanoma (e.g.,metastatic melanoma), an epithelial or epithelial-derived tumor,pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer,non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g.,lymphocytic leukemia, chronic myelogenous leukemia, acute myeloidleukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma(hepatocellular carcinoma or HCC), sarcoma, B-cell malignancy, breastcancer (for example, estrogen receptor positive (ER+), estrogen receptornegative (ER−), Her2 positive (Her2+), Her2 negative (Her2−), or acombination thereof, e.g., ER+/Her2+, ER+/Her2−, ER−/Her2+, orER−/Her2−; or “triple negative” breast cancer which is estrogenreceptor-negative, progesterone receptor-negative, and HER2-negative),ovarian cancer, colorectal cancer, glioma (e.g., astrocytoma,oligodendroglioma, ependymoma, or a choroid plexus papilloma),glioblastoma multiforme (e.g., giant cell gliobastoma or a gliosarcoma),meningioma, pituitary adenoma, vestibular schwannoma, primary CNSlymphoma, primitive neuroectodermal tumor (medulloblastoma), kidneycancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer,esophageal cancer, brain cancer, head and neck cancers, cervical cancer,testicular cancer, thyroid cancer, stomach cancer, virus-induced tumorssuch as, for example, papilloma virus-induced carcinomas (e.g., cervicalcarcinoma, cervical cancer), adenocarcinomas, herpes virus-inducedtumors (e.g., Burkitt's lymphoma, EBV-induced B-cell lymphoma),hepatitis B-induced tumors (hepatocellular carcinomas), HTLV-1-inducedand HTLV-2-induced lymphomas, acoustic neuroma, lung cancers (e.g., lungcarcinoma, bronchial carcinoma), small-cell lung carcinomas, pharyngealcancer, anal carcinoma, glioblastoma, rectal carcinoma, astrocytoma,brain tumors, retinoblastoma, basalioma, brain metastases,medulloblastomas, vaginal cancer, pancreatic cancer, testicular cancer,Hodgkin's syndrome, meningiomas, Schneeberger disease, hypophysis tumor,Mycosis fungoides, carcinoids, neurinoma, spinalioma, Burkitt'slymphoma, laryngeal cancer, renal cancer, thymoma, corpus carcinoma,bone cancer, non-Hodgkin's lymphomas, urethral cancer, CUP syndrome,head/neck tumors, oligodendroglioma, vulval cancer, intestinal cancer,colon carcinoma, oesophageal cancer (e.g., oesophageal carcinoma), wartinvolvement, tumors of the small intestine, craniopharyngeomas, ovariancarcinoma, genital tumors, ovarian cancer (e.g., ovarian carcinoma),pancreatic cancer (e.g., pancreatic carcinoma), endometrial carcinoma,liver metastases, penile cancer, tongue cancer, gall bladder cancer,leukaemia, plasmocytoma, and lid tumor.

In some embodiments, as noted above, the cancer or tumor is a metastaticcancer, for example, a metastatic cancer that expresses NRP2 and/orNRP2B. Further to the above cancers, exemplary metastatic cancersinclude, without limitation, bladder cancers which have metastasized tothe bone, liver, and/or lungs; breast cancers which have metastasized tothe bone, brain, liver, and/or lungs; colorectal cancers which havemetastasized to the liver, lungs, and/or peritoneum; kidney cancerswhich have metastasized to the adrenal glands, bone, brain, liver,and/or lungs; lung cancers which have metastasized to the adrenalglands, bone, brain, liver, and/or other lung sites; melanomas whichhave metastasized to the bone, brain, liver, lung, and/or skin/muscle;ovarian cancers which have metastasized to the liver, lung, and/orperitoneum; pancreatic cancers which have metastasized to the liver,lung, and/or peritoneum; prostate cancers which have metastasized to theadrenal glands, bone, liver, and/or lungs; stomach cancers which havemetastasized to the liver, lung, and/or peritoneum; thyroid cancerswhich have metastasized to the bone, liver, and/or lungs; and uterinecancers which have metastasized to the bone, liver, lung, peritoneum,and/or vagina; among others.

In some embodiments, for example, where the cancer immunotherapy agentis a PD-1 or PD-L1 antagonist or inhibitor, the subject has one or morebiomarkers (e.g., increased PD-1 or PD-L1 levels in cells such as cancercells or cancer-specific CTLs) that make the suitable for PD-1 or PD-L1inhibitor therapy. For instance, in some embodiments, the subject hasincreased fractions of programmed cell death 1 high/cytotoxic Tlymphocyte-associated protein 4 high (e.g., PD-1^(hi)CTLA-4^(hi) cellswithin a tumor-infiltrating CD8+ T cell subset (see, e.g., Daud et al.,J Clin Invest. 126:3447-3452, 2016). As another example, in someembodiments, the subject has increased levels of Bim (B cell lymphoma2-interacting (Bcl2-interacting) mediator) in circulating tumor-reactive(e.g., PD-1⁺ CD11a^(hi)CD8⁺)T cells, and optionally has metastaticmelanoma (see, e.g., Dronca et al., JCI Insight. May 5; 1(6): e86014,2016).

Certain specific combinations include an anti-NRP2 antibody and a PD-L1antagonist or inhibitor, for example, atezolizumab (MPDL3280A), avelumab(MSB0010718C), and durvalumab (MEDI4736), for treating a cancer selectedfrom one or more of colorectal cancer, melanoma, breast cancer,non-small-cell lung carcinoma, bladder cancer, and renal cell carcinoma.

Some specific combinations include an anti-NRP2 antibody and a PD-1antagonist, for example, nivolumab, for treating a cancer selected fromone or more of Hodgkin's lymphoma, melanoma, non-small cell lung cancer,hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer.

Particular specific combinations include an anti-NRP2 antibody and aPD-1 antagonist, for example, pembrolizumab, for treating a cancerselected from one or more of melanoma, non-small cell lung cancer, smallcell lung cancer, head and neck cancer, and urothelial cancer.

Certain specific combinations include an anti-NRP2 antibody and a CTLA-4antagonist, for example, ipilimumab and tremelimumab, for treating acancer selected from one or more of melanoma, prostate cancer, lungcancer, and bladder cancer.

Some specific combinations include an anti-NRP2 antibody and an IDOantagonist, for example, indoximod (NLG-8189), 1-methyl-tryptophan(1MT), β-Carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinicacid, or epacadostat, for treating a cancer selected from one or more ofmetastatic breast cancer and brain cancer optionally GlioblastomaMultiforme, glioma, gliosarcoma or malignant brain tumor.

Certain specific combinations include an anti-NRP2 antibody and thecytokine INF-α for treating melanoma, Kaposi sarcoma, and hematologiccancers. Also included is the combination of an anti-NRP2 antibody andIL-2 (e.g., Aldesleukin) for treating metastatic kidney cancer ormetastatic melanoma.

Some specific combinations include an anti-NRP2 antibody and a T-cellbased adoptive immunotherapy, for example, comprising CAR-modifiedT-cells targeted against CD-19, for treating hematological cancers suchas acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia(CLL), and B-cell neoplasms (see, e.g., Maude et al., 2015, supra;Lorentzen and Straten, Scand J Immunol. 82:307-19, 2015; and Ramos etal., Cancer J. 20:112-118, 2014).

The methods for treating cancers can be combined with other therapeuticmodalities. For example, a combination therapy described herein can beadministered to a subject before, during, or after other therapeuticinterventions, including symptomatic care, radiotherapy, surgery,transplantation, hormone therapy, photodynamic therapy, antibiotictherapy, or any combination thereof. Symptomatic care includesadministration of corticosteroids, to reduce cerebral edema, headaches,cognitive dysfunction, and emesis, and administration ofanti-convulsants, to reduce seizures. Radiotherapy includes whole-brainirradiation, fractionated radiotherapy, and radiosurgery, such asstereotactic radiosurgery, which can be further combined withtraditional surgery.

Methods for identifying subjects with one or more of the diseases orconditions (for example, NRP2-associated diseases or conditions)described herein are known in the art.

For in vivo use, as noted above, for the treatment of human or non-humanmammalian disease or testing, the agents described herein are generallyincorporated into one or more therapeutic or pharmaceutical compositionsprior to administration, including veterinary therapeutic compositions.

Thus, certain embodiments relate to therapeutic compositions thatcomprise at least one antibody or antigen-binding fragment thereof thatspecifically binds to a human NRP2 polypeptide, as described herein. Insome instances, a therapeutic or pharmaceutical composition comprisesone or more of the agents described herein in combination with apharmaceutically- or physiologically-acceptable carrier or excipient.Certain therapeutic compositions further comprise at least one cancerimmunotherapy agent, as described herein.

Some therapeutic compositions comprise (and certain methods utilize)only one anti-NRP2 antibody or antigen-binding fragment thereof. Certaintherapeutic compositions comprise (and certain methods utilize) amixture of at least two, three, four, or five different anti-NRP2antibodies or antigen-binding fragments thereof.

For instance, certain therapeutic compositions comprise at least twoanti-NRP2 antibodies, including a first antibody or antigen-bindingfragment thereof that specifically binds to at least one first epitopeof a human NRP2 polypeptide, and a second antibody or antigen-bindingfragment thereof that specifically binds to at least one second epitopeof a human NRP2 polypeptide, wherein the at least one first epitopediffers from the at least one second epitope. In some embodiments, thefirst and the second antibody or antigen-binding fragment thereofspecifically and non-competitively bind to the same domain of the NRP2polypeptide.

In some embodiments, the first and the second antibody orantigen-binding fragment thereof specifically and non-competitively bindto different domains of the NRP2 polypeptide.

In certain embodiments, the first antibody or antigen-binding fragmentis an antagonistic antibody (e.g., interferes with one or more signalingactivities of the HRS/NRP2 binding interaction), and the second antibodyor antigen-binding fragment is an agonist antibody (e.g., mimics oragonizes one or more signaling activities of the HRS/NRP2 bindinginteraction). In some embodiments, the first antibody antagonizes thebinding/signaling activity between the NRP2 polypeptide and the at leastone NRP2 ligand. In certain embodiments, the second antibody orantigen-binding fragment thereof agonizes or enhances thebinding/signaling activity between the NRP2 polypeptide and at least oneNRP2 ligand.

In some embodiments, the first and the second antibody orantigen-binding fragments thereof are both blocking antibodies, forexample, for at least two different NRP2 ligands. In some embodiments,the first and the second antibody or antigen-binding fragments thereofare both partial-blocking antibodies, for example, for at least twodifferent NRP2 ligands. In some instances, the first and the secondantibodies or antigen-binding fragments thereof are both non-blockingantibodies, for example, with respect to at least two different NRP2ligands.

In some instances, the first antibody or antigen-binding fragmentthereof is a blocking antibody and the second antibody orantigen-binding fragment thereof is a partial-blocking antibody. Incertain instances, the first antibody or antigen-binding fragmentthereof is a blocking antibody and the second antibody orantigen-binding fragment thereof is a non-blocking antibody.

In some embodiments, the first and the second antibodies orantigen-binding fragments thereof both comprise an IgG Fc domain withhigh effector function in humans, for example, an IgG1 or IgG3 Fcdomain. In some embodiments, the first and the second antibodies orantigen-binding fragments thereof comprise an IgG Fc domain with loweffector function in humans, for example, an IgG2 or IgG4 Fc domain.

In some instances, the first antibody or antigen-binding fragmentthereof comprises an IgG Fc domain with high effector function inhumans, for example, an IgG1 or IgG3 Fc domain, and the second antibodyor antigen-binding fragment thereof comprises an IgG Fc domain with loweffector function in humans, for example, an IgG2 or IgG4 Fc domain.

In particular embodiments, the therapeutic composition comprising theagents such as antibodies or other polypeptide agents (e.g., anti-NRP2antibodies) is substantially pure on a protein basis or a weight-weightbasis, for example, the composition has a purity of at least about 80%,85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis.

In some embodiments, the antibodies (e.g., anti-NRP2 antibodies) orother polypeptide agents provided herein do not form aggregates, have adesired solubility, and/or have an immunogenicity profile that issuitable for use in humans, as described herein and known in the art.Thus, in some embodiments, the therapeutic composition comprising apolypeptide agent (for example, an antibody such as an anti-NRP2antibody) is substantially aggregate-free. For example, certaincompositions comprise less than about 10% (on a protein basis) highmolecular weight aggregated proteins, or less than about 5% highmolecular weight aggregated proteins, or less than about 4% highmolecular weight aggregated proteins, or less than about 3% highmolecular weight aggregated proteins, or less than about 2% highmolecular weight aggregated proteins, or less than about 1% highmolecular weight aggregated proteins. Some compositions comprise apolypeptide agent (e.g., an antibody such as an anti-NRP2 antibody) thatis at least about 50%, about 60%, about 70%, about 80%, about 90% orabout 95% monodisperse with respect to its apparent molecular mass.

In some embodiments, polypeptide agents such as antibodies (e.g.,anti-NRP2 antibodies) are concentrated to about or at least about 0.1mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6, 0.7, 0.8, 0.9, 1mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9mg/ml, 10 mg/ml, 11, 12, 13, 14 or 15 mg/ml and are formulated forbiotherapeutic uses.

To prepare a therapeutic or pharmaceutical composition, an effective ordesired amount of one or more agents is mixed with any pharmaceuticalcarrier(s) or excipient known to those skilled in the art to be suitablefor the particular agent and/or mode of administration. A pharmaceuticalcarrier may be liquid, semi-liquid or solid. Solutions or suspensionsused for parenteral, intradermal, subcutaneous or topical applicationmay include, for example, a sterile diluent (such as water), salinesolution (e.g., phosphate buffered saline; PBS), fixed oil, polyethyleneglycol, glycerin, propylene glycol or other synthetic solvent;antimicrobial agents (such as benzyl alcohol and methyl parabens);antioxidants (such as ascorbic acid and sodium bisulfite) and chelatingagents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (suchas acetates, citrates and phosphates). If administered intravenously(e.g., by IV infusion), suitable carriers include physiological salineor phosphate buffered saline (PBS), and solutions containing thickeningand solubilizing agents, such as glucose, polyethylene glycol,polypropylene glycol and mixtures thereof.

Administration of agents described herein, in pure form or in anappropriate therapeutic or pharmaceutical composition, can be carriedout via any of the accepted modes of administration of agents forserving similar utilities. The therapeutic or pharmaceuticalcompositions can be prepared by combining an agent-containingcomposition with an appropriate physiologically acceptable carrier,diluent or excipient, and may be formulated into preparations in solid,semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants,gels, microspheres, and aerosols. In addition, other pharmaceuticallyactive ingredients (including other small molecules as describedelsewhere herein) and/or suitable excipients such as salts, buffers andstabilizers may, but need not, be present within the composition.

Administration may be achieved by a variety of different routes,including oral, parenteral, nasal, intravenous, intradermal,intramuscular, subcutaneous or topical. Preferred modes ofadministration depend upon the nature of the condition to be treated orprevented. Particular embodiments include administration by IV infusion.

Carriers can include, for example, pharmaceutically- orphysiologically-acceptable carriers, excipients, or stabilizers that arenon-toxic to the cell or mammal being exposed thereto at the dosages andconcentrations employed. Often the physiologically-acceptable carrier isan aqueous pH buffered solution. Examples of physiologically acceptablecarriers include buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as polysorbate 20 (TWEEN™) polyethylene glycol (PEG), andpoloxamers (PLURONICS™), and the like.

In some embodiments, one or more agents can be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate)microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980). The particle(s) or liposomes may further comprise othertherapeutic or diagnostic agents.

The precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by testing the compositions in model systems knownin the art and extrapolating therefrom. Controlled clinical trials mayalso be performed. Dosages may also vary with the severity of thecondition to be alleviated. A pharmaceutical composition is generallyformulated and administered to exert a therapeutically useful effectwhile minimizing undesirable side effects. The composition may beadministered one time, or may be divided into a number of smaller dosesto be administered at intervals of time. For any particular subject,specific dosage regimens may be adjusted over time according to theindividual need.

Typical routes of administering these and related therapeutic orpharmaceutical compositions thus include, without limitation, oral,topical, transdermal, inhalation, parenteral, sublingual, buccal,rectal, vaginal, and intranasal. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrasternal injection or infusion techniques. Therapeutic orpharmaceutical compositions according to certain embodiments of thepresent disclosure are formulated so as to allow the active ingredientscontained therein to be bioavailable upon administration of thecomposition to a subject or patient. Compositions that will beadministered to a subject or patient may take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container of a herein described agent in aerosol form may hold aplurality of dosage units. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington: The Science and Practice of Pharmacy, 20thEdition (Philadelphia College of Pharmacy and Science, 2000). Thecomposition to be administered will typically contain a therapeuticallyeffective amount of an agent described herein, for treatment of adisease or condition of interest.

A therapeutic or pharmaceutical composition may be in the form of asolid or liquid. In one embodiment, the carrier(s) are particulate, sothat the compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral oil, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration. When intended for oraladministration, the pharmaceutical composition is preferably in eithersolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid. Certain embodiments include sterile, injectable solutions.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like. Such a solid compositionwill typically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following may be present: binders such ascarboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gumtragacanth or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin; a flavoring agent such as peppermint,methyl salicylate or orange flavoring; and a coloring agent. When thepharmaceutical composition is in the form of a capsule, for example, agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil.

The therapeutic or pharmaceutical composition may be in the form of aliquid, for example, an elixir, syrup, solution, emulsion or suspension.The liquid may be for oral administration or for delivery by injection,as two examples. When intended for oral administration, preferredcomposition contain, in addition to the present compounds, one or moreof a sweetening agent, preservatives, dye/colorant and flavor enhancer.In a composition intended to be administered by injection, one or moreof a surfactant, preservative, wetting agent, dispersing agent,suspending agent, buffer, stabilizer and isotonic agent may be included.

The liquid therapeutic or pharmaceutical compositions, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid therapeutic or pharmaceutical composition intended for eitherparenteral or oral administration should contain an amount of an agentsuch that a suitable dosage will be obtained. Typically, this amount isat least 0.01% of the agent of interest in the composition. Whenintended for oral administration, this amount may be varied to bebetween 0.1 and about 70% of the weight of the composition. Certain oraltherapeutic or pharmaceutical compositions contain between about 4% andabout 75% of the agent of interest. In certain embodiments, therapeuticor pharmaceutical compositions and preparations are prepared so that aparenteral dosage unit contains between 0.01 to 10% by weight of theagent of interest prior to dilution.

The therapeutic or pharmaceutical compositions may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in atherapeutic or pharmaceutical composition for topical administration. Ifintended for transdermal administration, the composition may include atransdermal patch or iontophoresis device.

The therapeutic or pharmaceutical compositions may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter, and polyethylene glycol.

The therapeutic or pharmaceutical composition may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule. The therapeutic or pharmaceutical compositions in solid orliquid form may include a component that binds to agent and therebyassists in the delivery of the compound. Suitable components that mayact in this capacity include monoclonal or polyclonal antibodies, one ormore proteins or a liposome.

The therapeutic or pharmaceutical composition may consist essentially ofdosage units that can be administered as an aerosol. The term aerosol isused to denote a variety of systems ranging from those of colloidalnature to systems consisting of pressurized packages. Delivery may be bya liquefied or compressed gas or by a suitable pump system thatdispenses the active ingredients. Aerosols may be delivered in singlephase, bi-phasic, or tri-phasic systems in order to deliver the activeingredient(s). Delivery of the aerosol includes the necessary container,activators, valves, subcontainers, and the like, which together may forma kit. One of ordinary skill in the art, without undue experimentationmay determine preferred aerosols.

The compositions described herein may be prepared with carriers thatprotect the agents against rapid elimination from the body, such as timerelease formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, polyorthoesters, polylactic acid and others known to those ofordinary skill in the art.

The therapeutic or pharmaceutical compositions may be prepared bymethodology well known in the pharmaceutical art. For example, atherapeutic or pharmaceutical composition intended to be administered byinjection may comprise one or more of salts, buffers and/or stabilizers,with sterile, distilled water so as to form a solution. A surfactant maybe added to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact withthe agent so as to facilitate dissolution or homogeneous suspension ofthe agent in the aqueous delivery system.

The therapeutic or pharmaceutical or IVIG compositions may beadministered in a therapeutically effective amount, which will varydepending upon a variety of factors including the activity of thespecific compound employed; the metabolic stability and length of actionof the compound; the age, body weight, general health, sex, and diet ofthe subject; the mode and time of administration; the rate of excretion;the drug combination; the severity of the particular disorder orcondition; and the subject undergoing therapy. In some instances, atherapeutically effective daily dose is (for a 70 kg mammal) from about0.001 mg/kg (i.e., ^(˜)0.07 mg) to about 100 mg/kg (i.e., ^(˜)7.0 g);preferably a therapeutically effective dose is (for a 70 kg mammal) fromabout 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., ^(˜)3.5 g);more preferably a therapeutically effective dose is (for a 70 kg mammal)from about 1 mg/kg (i.e., ^(˜)70 mg) to about 25 mg/kg (i.e., ^(˜)1.75g). In some embodiments, the therapeutically effective dose isadministered on a weekly, bi-weekly, or monthly basis. In specificembodiments, the therapeutically effective dose is administered on aweekly, bi-weekly, or monthly basis, for example, at a dose of about1-10 or 1-5 mg/kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg.

The combination therapies described herein may include administration ofa single pharmaceutical dosage formulation, which contains an anti-NRP2antibody and an additional therapeutic agent (e.g., immunotherapy agent,chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor),as well as administration of compositions comprising an anti-NRP2antibody and an additional therapeutic agent in its own separatepharmaceutical dosage formulation. For example, an anti-NRP2 antibody asdescribed herein and additional therapeutic agent can be administered tothe subject together in a single oral dosage composition such as atablet or capsule, or each agent administered in separate oral dosageformulations. Similarly, an anti-NRP2 antibody as described herein andadditional therapeutic agent can be administered to the subject togetherin a single parenteral dosage composition such as in a saline solutionor other physiologically acceptable solution, or each agent administeredin separate parenteral dosage formulations. As another example, forcell-based therapies, an anti-NRP2 antibody can be mixed with the cellsprior to administration, administered as part of a separate composition,or both. Where separate dosage formulations are used, the compositionscan be administered at essentially the same time, i.e., concurrently, orat separately staggered times, i.e., sequentially and in any order;combination therapy is understood to include all these regimens.

Also included are patient care kits, comprising (a) at least oneantibody or antigen-binding fragment thereof that specifically binds toa human neuropilin 2 (NRP2) polypeptide (an anti-NRP2 antibody), asdescribed herein; and optionally (b) at least one additional therapeuticagent (e.g., immunotherapy agent, chemotherapeutic agent, hormonaltherapeutic agent, kinase inhibitor). In certain kits, (a) and (b) arein separate therapeutic compositions. In some kits, (a) and (b) are inthe same therapeutic composition.

The kits herein may also include a one or more additional therapeuticagents or other components suitable or desired for the indication beingtreated, or for the desired diagnostic application. The kits herein canalso include one or more syringes or other components necessary ordesired to facilitate an intended mode of delivery (e.g., stents,implantable depots, etc.).

In some embodiments, a patient care kit contains separate containers,dividers, or compartments for the composition(s) and informationalmaterial(s). For example, the composition(s) can be contained in abottle, vial, or syringe, and the informational material(s) can becontained in association with the container. In some embodiments, theseparate elements of the kit are contained within a single, undividedcontainer. For example, the composition is contained in a bottle, vialor syringe that has attached thereto the informational material in theform of a label. In some embodiments, the kit includes a plurality(e.g., a pack) of individual containers, each containing one or moreunit dosage forms (e.g., a dosage form described herein) of an anti-NRP2antibody and optionally at least one additional therapeutic agent. Forexample, the kit includes a plurality of syringes, ampules, foilpackets, or blister packs, each containing a single unit dose of ananti-NRP2 antibody and optionally at least one additional therapeuticagent. The containers of the kits can be air tight, waterproof (e.g.,impermeable to changes in moisture or evaporation), and/or light-tight.

The patient care kit optionally includes a device suitable foradministration of the composition, e.g., a syringe, inhalant, dropper(e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or anysuch delivery device. In some embodiments, the device is an implantabledevice that dispenses metered doses of the agent(s). Also included aremethods of providing a kit, e.g., by combining the components describedherein.

Bioassays and Analytical Assays for Drug Release Assays and ProductSpecifications, Diagnostics, and Reagents

Also included are bioassays that relate to anti-NRP2 antibodies andrelated agents such as therapeutic and diagnostic reagents. Examplesinclude bioassays and analytical assays that measure purity, biologicalactivity, affinity, solubility, pH, endotoxin levels, among others, manyof which are described herein. Also included are assays that establishdose response curves and/or provide one or more bases for comparisonbetween different batches of antibody. Batch comparisons can be based onany one or more of chemical characterization, biologicalcharacterization, and clinical characterization. Also included aremethods of evaluating the potency, stability, pharmacokinetics, andimmunogenicity of a selected antibody. Among other uses, these and othermethods can be used for lot releasing testing of biologic or chemicalagents, including anti-NRP2 antibodies, described herein.

Certain embodiments include the use of bioaffinity assays. Such assayscan be used to assess the binding affinity, for example, between ananti-NRP2 antibody and at least one NRP2 ligand (for example, an NRP2ligand from Table N2 or Table N3 and/or an HRS polypeptide from TableH1), including its ability to interfere with the interaction between ahuman NRP2 polypeptide and the at least one NRP2 ligand, or othercellular binding partner. Certain exemplary binding affinity assays mayutilize ELISA assays, and other immunoassays as described herein andknown in the art. Certain assays utilize high-performance receptorbinding chromatography (see, e.g., Roswall et al., Biologicals.24:25-39, 1996). Other exemplary binding affinity assays may utilizesurface plasmon resonance (SPR)-based technologies. Examples includeBIACore technologies, certain of which integrate SPR technology with amicrofluidics system to monitor molecular interactions in real time atconcentrations ranging from pM to mM. Also included are KINEXA™ assays,which provide accurate measurements of binding specificity, bindingaffinity, and binding kinetics/rate constants.

Certain embodiments relate to immunoassays for evaluating or optimizingthe immunogenicity of anti-NRP2 antibodies. Examples include ex vivohuman cellular assays and in vitro immuno-enzymatic assays to provideuseful information on the immunogenic potential of a therapeuticprotein. Ex vivo cell-response assays can be used, for example, toreproduce the cellular co-operation between antigen-presenting cells(APCs) and T-cells, and thereby measure T-cells activation after contactwith a protein of interest. Certain in vitro enzymatic assays mayutilize a collection of recombinant HLA-DR molecules that cover asignificant portion of a relevant human population, and may includeautomated immuno-enzymatic assays for testing the binding of peptides(stemming from the fragmentation of the therapeutic protein) with theHLA-DR molecules. Also included are methods of reducing theimmunogenicity of a selected protein, such as by using these and relatedmethods to identify and then remove or alter one or more T-cell epitopesfrom an anti-NRP2 antibody.

Also included are biological release assays (e.g., cell-based assays)for measuring parameters such as specific biological activities,including non-canonical biological activities, and cytotoxicity. Certainspecific biological assays include, for example, cell-based assays thatutilize a cellular binding partner (e.g., cell-surface receptor (forexample a NRP2 polypeptide and/or at least one NRP2 ligand (for example,an NRP2 ligand from Table N2 or Table N3) presented on the cellsurface), which is either endogenously, or recombinantly expressed onthe cell surface), which is functionally coupled to a readout, such as afluorescent or luminescent indicator of NRP2 or NRP2 ligand binding, orfunctional activity, as described herein.

For instance, specific embodiments include a cell that eitherendogenously or recombinantly expresses a human NRP2 polypeptide on thecell surface, which allows assessment of the ability of anti-NRP2antibody to bind NRP2. In some embodiments, the anti-NRP2 antibodyand/or the NRP2 polypeptide is/are functionally coupled to a readout orindicator, such as a fluorescent or luminescent indicator to measure thebinding and/or biological activity of the NRP2 polypeptide. In someembodiments, the cell also expresses at least one NRP2 ligand (forexample, an NRP2 ligand from Table N2 or Table N3 and/or an HRSpolypeptide from Table H1), wherein the at least one NRP2 ligand iscoupled to a readout or indicator, such as a fluorescent or luminescentindicator of binding and/or biological activity of the at least one NRP2ligand.

Also included are in vivo biological assays to characterize thepharmacokinetics of an anti-NRP2 antibody, typically utilizingengineered, or wild type mice, rat, monkey or other mammal (see, e.g.,Lee et al., The Journal of Pharmacology. 281:1431-1439, 1997). Examplesof cytotoxicity-based biological assays include release assays (e.g.,chromium or europium release assays to measure apoptosis; see, e.g., vonZons et al., Clin Diagn Lab Immunol. 4:202-207, 1997), among others,which can assess the cytotoxicity anti-NRP2 antibodies, whether forestablishing dose response curves, batch testing, or other propertiesrelated to approval by various regulatory agencies, such as the Food andDrug Administration (FDA).

Also included are assays for evaluating the effects of an anti-NRP2antibody on immune cells. Examples include an assay system, comprisingan activated population of T-cells and at least one anti-NRP2 antibody,wherein the at least one anti-NRP2 antibody interferes with binding ofNRP2 to at least one NRP2 ligand (for example, an NRP2 ligand from TableN2 or Table N3 and/or an HRS polypeptide from Table H1).

Certain embodiments include an assay system, comprising a singlemonoclonal anti-NRP2 antibody and at least one human NRP2 polypeptide,wherein the anti-NRP2 antibody binds to the NRP2 polypeptide. In someinstances, the at least one antibody comprises an IgG4 Fc domain.

Also included are testing material(s), comprising a purified NRP2polypeptide, wherein said purified NRP2 polypeptide is bound to a solidsubstrate in a manner that enables antibody binding detection.

Such assays and materials can be used, for example, to develop a doseresponse curve for a selected anti-NRP2 antibody, and/or to compare thedose response curve of different batches of proteins or other agents. Adose-response curve is an X-Y graph that relates the magnitude of astressor to the response of a receptor, such as an NRP2-NRP2 ligand (forexample, an NRP2 ligand from Table N2 or Table N3 and/or an HRSpolypeptide from Table H1) interaction; the response may be aphysiological or biochemical response, such as a non-canonicalbiological activity in a cell in vitro or in a cell or tissue in vivo, atherapeutically effective amount as measured in vivo (e.g., as measuredby EC₅₀), or death, whether measured in vitro or in vivo (e.g., celldeath, organismal death). Death is usually indicated as an LD₅₀, astatistically-derived dose that is lethal to 50% of a modeledpopulation, though it can be indicated by LC₀₁ (lethal dose for 1% ofthe animal test population), LC₁₀₀ (lethal dose for 100% of the animaltest population), or LC_(LO) (lowest dose causing lethality). Almost anydesired effect or endpoint can be characterized in this manner.

The measured dose of a response curve is typically plotted on the X axisand the response is plotted on the Y axis. More typically, the logarithmof the dose is plotted on the X axis, most often generating a sigmoidalcurve with the steepest portion in the middle. The No Observable EffectLevel (NOEL) refers to the lowest experimental dose for which nomeasurable effect is observed, and the threshold dose refers to thefirst point along the graph that indicates a response above zero. As ageneral rule, stronger drugs generate steeper dose response curves. Formany drugs, the desired effects are found at doses slightly greater thanthe threshold dose, often because lower doses are relatively ineffectiveand higher doses lead to undesired side effects. For in vivo generateddose response curves, a curve can be characterized by values such asμg/kg, mg/kg, or g/kg of body-weight, if desired.

For batch comparisons, it can be useful to calculate the coefficient ofvariation (CV) between different dose response curves of differentbatches (e.g., between different batches of anti-NRP2 antibody), in partbecause the CV allows comparison between data sets with different unitsor different means. For instance, in certain exemplary embodiments, twoor three or more different batches of anti-NRP2 antibodies or otheragents have a CV between them of less than about 30%, 20%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% for a 4, 5, 6,7, or 8 point dose curve. In certain embodiments, the dose responsecurve is measured in a cell-based assay, and its readout relates to anincrease or a decrease in a selected activity of an anti-NRP2 antibody.In certain embodiments, the dose response curve is measured in a cellrelease assay or animal model (e.g., mouse model), and its readoutrelates to cell death or animal death. Other variations will be apparentto persons skilled in the art.

Expression and Purification Systems

Certain embodiments include methods and related compositions forexpressing and purifying an anti-NRP2 antibody or otherpolypeptide-based agent described herein. Such recombinant anti-NRP2antibodies can be conveniently prepared using standard protocols asdescribed for example in Sambrook, et al., (1989, supra), in particularSections 16 and 17; Ausubel et al., (1994, supra), in particularChapters 10 and 16; and Coligan et al., Current Protocols in ProteinScience (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5and 6. As one general example, anti-NRP2 antibodies may be prepared by aprocedure including one or more of the steps of: (a) preparing aconstruct comprising a polynucleotide sequences that encode an anti-NRP2antibody heavy and light chain and that are operably linked to aregulatory element; (b) introducing the constructs into a host cell; (c)culturing the host cell to express an anti-NRP2 antibody; and (d)isolating an anti-NRP2 antibody from the host cell.

Anti-NRP2 antibody polynucleotides are described elsewhere herein. Inorder to express a desired polypeptide, a nucleotide sequence encodingan anti-NRP2 antibody, or a functional equivalent, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems, including mammalian cell and more specifically human cellsystems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, vectors which direct highlevel expression of fusion proteins that are readily purified may beused. Such vectors include, but are not limited to, the multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the sequence encoding the polypeptide of interest may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264:5503 5509 (1989)); and the like. pGEX Vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

Certain embodiments may employ E. coli-based expression systems (see,e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146,2008). These and related embodiments may rely partially or totally onligation-independent cloning (LIC) to produce a suitable expressionvector. In specific embodiments, protein expression may be controlled bya T7 RNA polymerase (e.g., pET vector series). These and relatedembodiments may utilize the expression host strain BL21(DE3), a λDE3lysogen of BL21 that supports T7-mediated expression and is deficient inIon and ompT proteases for improved target protein stability. Alsoincluded are expression host strains carrying plasmids encoding tRNAsrarely used in E. coli, such as ROSETTA™ (DE3) and Rosetta 2 (DE3)strains. Cell lysis and sample handling may also be improved usingreagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER®Protein Extraction Reagent. For cell culture, auto-inducing media canimprove the efficiency of many expression systems, includinghigh-throughput expression systems. Media of this type (e.g., OVERNIGHTEXPRESS™ Autoinduction System) gradually elicit protein expressionthrough metabolic shift without the addition of artificial inducingagents such as IPTG. Particular embodiments employ hexahistidine tags(such as those sold under the trademark HIS•TAG® fusions), followed byimmobilized metal affinity chromatography (IMAC) purification, orrelated techniques. In certain aspects, however, clinical grade proteinscan be isolated from E. coli inclusion bodies, without or without theuse of affinity tags (see, e.g., Shimp et al., Protein Expr Purif.50:58-67, 2006). As a further example, certain embodiments may employ acold-shock induced E. coli high-yield production system, becauseover-expression of proteins in Escherichia coli at low temperatureimproves their solubility and stability (see, e.g., Qing et al., NatureBiotechnology. 22:877-882, 2004).

Also included are high-density bacterial fermentation systems. Forexample, high cell density cultivation of Ralstonia eutropha allowsprotein production at cell densities of over 150 g/L, and the expressionof recombinant proteins at titers exceeding 10 g/L.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al., Methods Enzymol. 153:516-544 (1987). Also included arePichia pandoris expression systems (see, e.g., Li et al., NatureBiotechnology. 24, 210-215, 2006; and Hamilton et al., Science,301:1244, 2003). Certain embodiments include yeast systems that areengineered to selectively glycosylate proteins, including yeast thathave humanized N-glycosylation pathways, among others (see, e.g.,Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., NatureReviews Microbiol. 3:119-28, 2005; and Gerngross et al.,Nature-Biotechnology. 22:1409-1414, 2004; U.S. Pat. Nos. 7,629,163;7,326,681; and 7,029,872). Merely by way of example, recombinant yeastcultures can be grown in Fernbach Flasks or 15L, 50L, 100L, and 200Lfermentors, among others.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw Hill,Yearbook of Science and Technology, pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia cells. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusiacells in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)). Alsoincluded are baculovirus expression systems, including those thatutilize SF9, SF21, and T. ni cells (see, e.g., Murphy and Piwnica-Worms,Curr Protoc Protein Sci. Chapter 5: Unit 5.4, 2001). Insect systems canprovide post-translation modifications that are similar to mammaliansystems.

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Examples of useful mammalian host cell lines include monkey kidney CV1line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidneyline (293 or 293 cells sub-cloned for growth in suspension culture,Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells(BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNASUSA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B. K. C Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.Certain preferred mammalian cell expression systems include CHO andHEK293-cell based expression systems. Mammalian expression systems canutilize attached cell lines, for example, in T-flasks, roller bottles,or cell factories, or suspension cultures, for example, in 1L and 5Lspinners, 5L, 14L, 40L, 100L and 200L stir tank bioreactors, or 20/50Land 100/200L WAVE bioreactors, among others known in the art.

Also included is the cell-free expression of proteins. These and relatedembodiments typically utilize purified RNA polymerase, ribosomes, tRNAand ribonucleotides; these reagents may be produced by extraction fromcells or from a cell-based expression system.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, post-translationalmodifications such as acetylation, carboxylation, glycosylation,phosphorylation, lipidation, and acylation. Post-translationalprocessing which cleaves a “prepro” form of the protein may also be usedto facilitate correct insertion, folding and/or function. Different hostcells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition tobacterial cells, which have or even lack specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for about 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type. Transientproduction, such as by transient transfection or infection, can also beemployed. Exemplary mammalian expression systems that are suitable fortransient production include HEK293 and CHO-based systems.

Any number of selection systems may be used to recover transformed ortransduced cell lines. These include, but are not limited to, the herpessimplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977))and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823(1990)) genes which can be employed in tk- or aprt-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of visible markers hasgained popularity with such markers as green fluorescent protein (GFP)and other fluorescent proteins (e.g., RFP, YFP), anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (see, e.g., Rhodes et al.,Methods Mol. Biol. 55:121-131 (1995)).

Also included are high-throughput protein production systems, ormicro-production systems. Certain aspects may utilize, for example,hexa-histidine fusion tags for protein expression and purification onmetal chelate-modified slide surfaces or MagneHis Ni-Particles (see,e.g., Kwon et al., BMC Biotechnol. 9:72, 2009; and Lin et al., MethodsMol Biol. 498:129-41, 2009)). Also included are high-throughputcell-free protein expression systems (see, e.g., Sitaraman et al.,Methods Mol Biol. 498:229-44, 2009). These and related embodiments canbe used, for example, to generate microarrays of anti-NRP2 antibodieswhich can then be used for screening libraries to identify antibodiesand antigen-binding domains that interact with the NRP2 polypeptide(s)of interest.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using binding agents or antibodies suchas polyclonal or monoclonal antibodies specific for the product, areknown in the art. Examples include enzyme-linked immunosorbent assay(ELISA), western immunoblots, radioimmunoassays (RIA), and fluorescenceactivated cell sorting (FACS). These and other assays are described,among other places, in Hampton et al., Serological Methods, a LaboratoryManual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. Certain specific embodiments utilizeserum free cell expression systems. Examples include HEK293 cells andCHO cells that can grown on serum free medium (see, e.g., Rosser et al.,Protein Expr. Purif. 40:237-43, 2005; and U.S. Pat. No. 6,210,922).

An antibody, or antigen-binding fragment thereof, produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides maybe designed to contain signal sequences which direct secretion of theencoded polypeptide through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encoding apolypeptide of interest to nucleotide sequence encoding a polypeptidedomain which will facilitate purification and/or detection of solubleproteins. Examples of such domains include cleavable and non-cleavableaffinity purification and epitope tags such as avidin, FLAG tags,poly-histidine tags (e.g., 6×His), cMyc tags, V5-tags, glutathioneS-transferase (GST) tags, and others.

The protein produced by a recombinant cell can be purified andcharacterized according to a variety of techniques known in the art.Exemplary systems for performing protein purification and analyzingprotein purity include fast protein liquid chromatography (FPLC) (e.g.,AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography(HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries forpurification include ion exchange chromatography (e.g., Q, S), sizeexclusion chromatography, salt gradients, affinity purification (e.g.,Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration,reverse-phase, ceramic HYPERD® ion exchange chromatography, andhydrophobic interaction columns (HIC), among others known in the art.Also included are analytical methods such as SDS-PAGE (e.g., coomassie,silver stain), immunoblot, Bradford, and ELISA, which may be utilizedduring any step of the production or purification process, typically tomeasure the purity of the protein composition.

Also included are methods of concentrating anti-NRP2 antibodies andantigen-binding fragments thereof, and composition comprisingconcentrated soluble proteins. In different aspects such concentratedsolutions of anti-NRP2 antibodies may comprise proteins at aconcentration of about 5 mg/mL; or about 8 mg/mL; or about 10 mg/mL;about 15 mg/mL; or about 20 mg/mL.

In some aspects, such compositions may be substantially monodisperse,meaning that an at least one anti-NRP2 antibody exists primarily (i.e.,at least about 90%, or greater) in one apparent molecular weight formwhen assessed for example, by size exclusion chromatography, dynamiclight scattering, or analytical ultracentrifugation.

In some aspects, such compositions have a purity (on a protein basis) ofat least about 90%, or in some aspects at least about 95% purity, or insome embodiments, at least 98% purity. Purity may be determined via anyroutine analytical method as known in the art.

In some aspects, such compositions have a high molecular weightaggregate content of less than about 10%, compared to the total amountof protein present, or in some embodiments such compositions have a highmolecular weight aggregate content of less than about 5%, or in someaspects such compositions have a high molecular weight aggregate contentof less than about 3%, or in some embodiments a high molecular weightaggregate content of less than about 1%. High molecular weight aggregatecontent may be determined via a variety of analytical techniquesincluding for example, by size exclusion chromatography, dynamic lightscattering, or analytical ultracentrifugation.

Examples of concentration approaches contemplated herein includelyophilization, which is typically employed when the solution containsfew soluble components other than the protein of interest.Lyophilization is often performed after HPLC run, and can remove most orall volatile components from the mixture. Also included areultrafiltration techniques, which typically employ one or more selectivepermeable membranes to concentrate a protein solution. The membraneallows water and small molecules to pass through and retains theprotein; the solution can be forced against the membrane by mechanicalpump, gas pressure, or centrifugation, among other techniques.

In certain embodiments, the reagents, anti-NRP2 antibodies, or relatedagents have a purity of at least about 90%, as measured according toroutine techniques in the art. In certain embodiments, such asdiagnostic compositions or certain therapeutic compositions, ananti-NRP2 antibody composition has a purity of at least about 95%. Inspecific embodiments, such as therapeutic or pharmaceuticalcompositions, an anti-NRP2 antibody composition has a purity of at leastabout 97% or 98% or 99%. In other embodiments, such as when being usedas reference or research reagents, anti-NRP2 antibodies can be of lesserpurity, and may have a purity of at least about 50%, 60%, 70%, or 80%.Purity can be measured overall or in relation to selected components,such as other proteins, e.g., purity on a protein basis.

Purified anti-NRP2 antibodies can also be characterized according totheir biological characteristics. Binding affinity and binding kineticscan be measured according to a variety of techniques known in the art,such as Biacore® and related technologies that utilize surface plasmonresonance (SPR), an optical phenomenon that enables detection ofunlabeled interactants in real time. SPR-based biosensors can be used indetermination of active concentration, screening and characterization interms of both affinity and kinetics. The presence or levels of one ormore canonical or non-canonical biological activities can be measuredaccording to cell-based assays, including those that utilize a cellularbinding partner of a selected anti-NRP2 antibody, which is functionallycoupled to a readout or indicator, such as a fluorescent or luminescentindicator of biological activity, as described herein.

In certain embodiments, as noted above, an anti-NRP2 antibodycomposition is substantially endotoxin free, including, for example,about 95% endotoxin free, preferably about 99% endotoxin free, and morepreferably about 99.99% endotoxin free. The presence of endotoxins canbe detected according to routine techniques in the art, as describedherein. In specific embodiments, an anti-NRP2 antibody composition ismade from a eukaryotic cell such as a mammalian or human cell insubstantially serum free media. In certain embodiments, as noted herein,an anti-NRP2 antibody composition has an endotoxin content of less thanabout 10 EU/mg of anti-NRP2 antibody, or less than about 5 EU/mg ofanti-NRP2 antibody, less than about 3 EU/mg of anti-NRP2 antibody, orless than about 1 EU/mg of anti-NRP2 antibody.

In certain embodiments, an anti-NRP2 antibody composition comprises lessthan about 10% wt/wt high molecular weight aggregates, or less thanabout 5% wt/wt high molecular weight aggregates, or less than about 2%wt/wt high molecular weight aggregates, or less than about or less thanabout 1% wt/wt high molecular weight aggregates.

Also included are protein-based analytical assays and methods, which canbe used to assess, for example, protein purity, size, solubility, anddegree of aggregation, among other characteristics. Protein purity canbe assessed a number of ways. For instance, purity can be assessed basedon primary structure, higher order structure, size, charge,hydrophobicity, and glycosylation. Examples of methods for assessingprimary structure include N- and C-terminal sequencing andpeptide-mapping (see, e.g., Allen et al., Biologicals. 24:255-275,1996)). Examples of methods for assessing higher order structure includecircular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta.1751:119-139, 2005), fluorescent spectroscopy (see, e.g., Meagher etal., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuteriumexchange kinetics, differential scanning calorimetry, NMR spectroscopy,immunoreactivity with conformationally sensitive antibodies. Higherorder structure can also be assessed as a function of a variety ofparameters such as pH, temperature, or added salts. Examples of methodsfor assessing protein characteristics such as size include analyticalultracentrifugation and size exclusion HPLC (SEC-HPLC), and exemplarymethods for measuring charge include ion-exchange chromatography andisolectric focusing. Hydrophobicity can be assessed, for example, byreverse-phase HPLC and hydrophobic interaction chromatography HPLC.Glycosylation can affect pharmacokinetics (e.g., clearance),conformation or stability, receptor binding, and protein function, andcan be assessed, for example, by mass spectrometry and nuclear magneticresonance (NMR) spectroscopy.

As noted above, certain embodiments include the use of SEC-HPLC toassess protein characteristics such as purity, size (e.g., sizehomogeneity) or degree of aggregation, and/or to purify proteins, amongother uses. SEC, also including gel-filtration chromatography (GFC) andgel-permeation chromatography (GPC), refers to a chromatographic methodin which molecules in solution are separated in a porous material basedon their size, or more specifically their hydrodynamic volume, diffusioncoefficient, and/or surface properties. The process is generally used toseparate biological molecules, and to determine molecular weights andmolecular weight distributions of polymers. Typically, a biological orprotein sample (such as a protein extract produced according to theprotein expression methods provided herein and known in the art) isloaded into a selected size-exclusion column with a defined stationaryphase (the porous material), preferably a phase that does not interactwith the proteins in the sample. In certain aspects, the stationaryphase is composed of inert particles packed into a densethree-dimensional matrix within a glass or steel column. The mobilephase can be pure water, an aqueous buffer, an organic solvent, or amixture thereof. The stationary-phase particles typically have smallpores and/or channels which only allow molecules below a certain size toenter. Large particles are therefore excluded from these pores andchannels, and their limited interaction with the stationary phase leadsthem to elute as a “totally-excluded” peak at the beginning of theexperiment. Smaller molecules, which can fit into the pores, are removedfrom the flowing mobile phase, and the time they spend immobilized inthe stationary-phase pores depends, in part, on how far into the poresthey penetrate. Their removal from the mobile phase flow causes them totake longer to elute from the column and results in a separation betweenthe particles based on differences in their size. A given size exclusioncolumn has a range of molecular weights that can be separated. Overall,molecules larger than the upper limit will not be trapped by thestationary phase, molecules smaller than the lower limit will completelyenter the solid phase and elute as a single band, and molecules withinthe range will elute at different rates, defined by their propertiessuch as hydrodynamic volume. For examples of these methods in practicewith pharmaceutical proteins, see Bruner et al., Journal ofPharmaceutical and Biomedical Analysis. 15: 1929-1935, 1997.

Protein purity for clinical applications is also discussed, for example,by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). Morerecent techniques for analyzing protein purity include, withoutlimitation, the LabChip GXII, an automated platform for rapid analysisof proteins and nucleic acids, which provides high throughput analysisof titer, sizing, and purity analysis of proteins. In certainnon-limiting embodiments, clinical grade proteins such as proteinfragments and antibodies can be obtained by utilizing a combination ofchromatographic materials in at least two orthogonal steps, among othermethods (see, e.g., Therapeutic Proteins: Methods and Protocols. Vol.308, Eds., Smales and James, Humana Press Inc., 2005). Typically,protein agents (e.g., anti-NRP2 antibodies, and antigen-bindingfragments) are substantially endotoxin-free, as measured according totechniques known in the art and described herein.

Protein solubility assays are also included. Such assays can beutilized, for example, to determine optimal growth and purificationconditions for recombinant production, to optimize the choice ofbuffer(s), and to optimize the choice of anti-NRP2 antibodies orvariants thereof. Solubility or aggregation can be evaluated accordingto a variety of parameters, including temperature, pH, salts, and thepresence or absence of other additives. Examples of solubility screeningassays include, without limitation, microplate-based methods ofmeasuring protein solubility using turbidity or other measure as an endpoint, high-throughput assays for analysis of the solubility of purifiedrecombinant proteins (see, e.g., Stenvall et al., Biochim Biophys Acta.1752:6-10, 2005), assays that use structural complementation of agenetic marker protein to monitor and measure protein folding andsolubility in vivo (see, e.g., Wigley et al., Nature Biotechnology.19:131-136, 2001), and electrochemical screening of recombinant proteinsolubility in Escherichia coli using scanning electrochemical microscopy(SECM) (see, e.g., Nagamine et al., Biotechnology and Bioengineering.96:1008-1013, 2006), among others. Anti-NRP2 antibodies with increasedsolubility (or reduced aggregation) can be identified or selected foraccording to routine techniques in the art, including simple in vivoassays for protein solubility (see, e.g., Maxwell et al., Protein Sci.8:1908-11, 1999).

Protein solubility and aggregation can also be measured by dynamic lightscattering techniques. Aggregation is a general term that encompassesseveral types of interactions or characteristics, includingsoluble/insoluble, covalent/noncovalent, reversible/irreversible, andnative/denatured interactions and characteristics. For proteintherapeutics, the presence of aggregates is typically consideredundesirable because of the concern that aggregates may cause animmunogenic reaction (e.g., small aggregates), or may cause adverseevents on administration (e.g., particulates). Dynamic light scatteringrefers to a technique that can be used to determine the sizedistribution profile of small particles in suspension or polymers suchas proteins in solution. This technique, also referred to as photoncorrelation spectroscopy (PCS) or quasi-elastic light scattering (QELS),uses scattered light to measure the rate of diffusion of the proteinparticles. Fluctuations of the scattering intensity can be observed dueto the Brownian motion of the molecules and particles in solution. Thismotion data can be conventionally processed to derive a sizedistribution for the sample, wherein the size is given by the Stokesradius or hydrodynamic radius of the protein particle. The hydrodynamicsize depends on both mass and shape (conformation). Dynamic scatteringcan detect the presence of very small amounts of aggregated protein(<0.01% by weight), even in samples that contain a large range ofmasses. It can also be used to compare the stability of differentformulations, including, for example, applications that rely onreal-time monitoring of changes at elevated temperatures. Accordingly,certain embodiments include the use of dynamic light scattering toanalyze the solubility and/or presence of aggregates in a sample thatcontains an anti-NRP2 antibody of the present disclosure.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this disclosure that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims. The following examples areprovided by way of illustration only and not by way of limitation. Thoseof skill in the art will readily recognize a variety of noncriticalparameters that could be changed or modified to yield essentiallysimilar results.

Example 1 Generation of Antibodies to Human Neuropilin 2

Antibody Generation.

The anti-NRP2 antibodies listed in Table E1 were generated by immunizingmice via an IP administration with 1×10⁶ Expi293 cells stably overexpressing human NRP2A variant 2 (Origene Technologies Cat#RC220706),(prepared as more fully described below) using standard methodologies.Titers were boosted via S.C. administration of 10 μg/mouse of thecorresponding recombinant NRP2 polypeptides listed in Table E2, usingeither IFA or Magic Mouse as the adjuvant. Mice were boosted every 2-3weeks and then screened for initial titer and specificity using the NRP2polypeptides listed in Table E2.

TABLE E1 Antibodies Antibody Immunogen # Clone Isotype Target domainaNRP2-1 1023 17f7 mIgG1k b2 aNRP2-2 1085 3f2 mIgG2ak c aNRP2-6 1024 8e2mIgG1k b1 aNRP2-7 1024 5h11 mIgG1k b1 aNRP2-8 1024 7g10 mIgG1k b1aNRP2-9 1024 9e7 mIgG1k b1 aNRP2-10 1024 1e3 mIgG1k b1 aNRP2-11 102413d7 mIgG2ak b2 aNRP2-12 1085 20f3 mIgG2ak c aNRP2-14 1024 18b8 mIgG1ka2 aNRP2-15 1024 19e8 mIgG2bk b1

TABLE E2 NRP2 Immunogens SEQ ID Immunogen # Sequence NO: NRP2 1023QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEPNQK 120 (23-595)-IVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCGNIAPPT FcIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK NRP2GSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIIL  22 (145-595)-QFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKT FcPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSISIS PGK NRP2 1085QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEPNQK  21 (23-855)-IVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCGNIAPPT FcIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSISISPGK

For all antibodies, spleens were isolated from immunized animals andfusion with mouse myeloma cells was performed to generate hybridomasusing standard techniques. Fusion, plating into 96-well plates, ELISAscreening of hybridomas, expansion and characterization of positivehybridomas (titer and isotype) and freezing of up to 15 hybridomas perantigen, was performed at The Scripps Research Institute (TSRI) Centerfor Antibody Development and Production. Antibody variable domainsequences were obtained using standard sequencing approaches performedat Lake Pharma, and are listed in Table A1.

Recombinant antibodies were produced from hybridoma cells afterexpansion and purified from conditioned medium starting at 2 weeks ofculture by flowing over a Protein A affinity column, eluting and storingin Phosphate Buffered Saline (1×PBS), pH 7.4. Each lot was tested forprotein concentration, purity, and endotoxin level. Purity by SDS-PAGEwas routinely >90%.

Example 2 Characterization of Anti-Human Neuropilin 2 Antibodies

Initial assessments of binding affinity as measured by enzyme-linkedimmunosorbent assay (ELISA), surface plasmon resonance (SPR), and flowcytometry (FACS) binding to cell lines expressing recombinant NRP2, aswell as the ability of the antibodies to block Fc-HRS(2-60), VEGF-Cbinding, or Semaphorin 3F are presented in Table E3.

Anti-NRP2 Antibody Binding and Affinity Measurements.

Surface plasmon resonance (SPR) methods were used to demonstrate bindingof anti-NRP2 antibodies to human NRP2 antigen and to measure bindingaffinities as summarized in Table E3. SPR experiments were conducted ona Bio-Rad ProteOn XPR36 Protein Interaction Array instrument. Goatanti-mouse antibody was immobilized on ProteOn GLC sensor chips by aminecoupling. Anti-NRP2 antibodies were subsequently flowed over andcaptured by the anti-mouse antibody. Human NRP2 antigen protein wasflowed over the captured antibody at varying concentrations (150, 50,16.67, 5.56, 1.85 nM). The sensor chip surface was regenerated betweeneach analyte run to remove anti-NRP2 antibodies and NRP2 protein. Datawas double referenced against a surface with no anti-NRP2 antibodycaptured (immobilized goat anti-mouse antibody only) and a buffer onlyblank. Affinity constants were derived by globally fitting sensograms toa Langmuir (1:1) interaction model in the ProteOn manager software. Foreach anti-NRP2 antibody, data from the multiple NRP2 concentrations wasfit as a single data set with dissociation rate constant (k_(d)),association rate constant (k_(a)) and R_(max) values as globalparameters. The reported binding affinity is the equilibriumdissociation constant (K_(D)) calculated from k_(d)/k_(a).

-   -   Running buffer: 50 mM HEPES, 300 mM NaCl, 5 mM CaCl₂, 0.005%        Tween-20, pH 7.4    -   Amine coupling: ProteOn Amine Coupling Kit (Bio-Rad #1762410)    -   Antibody coupling buffer: 10 mM sodium acetate pH 5.5    -   Immobilized antibody: AffiniPure Goat Anti-Mouse IgG, Fcγ        Fragment (Jackson ImmunoResearch #115-005-071)    -   Antigen: Human NRP2 (aa23-855) with C-terminal Avi-, Myc- and        His-tags    -   Regeneration buffer: 10 mM glycine pH 1.5

Ligand Blocking Studies.

SPR methods were used to demonstrate blocking of Fc-HRS(2-60), VEGF-Cand SEMS3F binding to NRP2 by anti-NRP2 antibodies as shown in Table E3.SPR experiments were conducted on a Bio-Rad ProteOn XPR36 ProteinInteraction Array instrument. Goat anti-mouse antibody was immobilizedon ProteOn GLC sensor chips by amine coupling. Anti-NRP2 antibodies wereflowed over and captured by the anti-mouse antibody. Human NRP2 proteinwas captured by the anti-NRP2 antibodies by flowing over at 400 nM.Either VEGF-C or SEMA3F-Fc was then flowed over the capturedNRP2:anti-NRP2 complex at 50 nM. The signal upon ligand addition wasreferenced against addition of buffer only. If subsequent binding of theligand (either VEGF-C or SEMA3F-Fc) was observed, that antibody wasdetermined to not block NRP2 ligand binding. Conversely, if no bindingof the ligand to the NRP2:anti-NRP2 complex was observed, that antibodywas determined to block binding of that NRP2 ligand. Flow cytometrybinding studies to 293 Epi NRP2 over expressing cells were conducted asdescribed in Example 4.

-   -   Running buffer: 50 mM HEPES, 300 mM NaCl, 5 mM CaCl₂, 0.005%        Tween-20, pH 7.4    -   Amine coupling: ProteOn Amine Coupling Kit (Bio-Rad #1762410)    -   Antibody coupling buffer: 10 mM sodium acetate pH 5.0    -   Immobilized antibody: AffiniPure Goat Anti-Mouse IgG, Fcγ        Fragment (Jackson ImmunoResearch #115-005-071)    -   Antigen: Human NRP2 (aa23-855) with C-terminal Avi-, Myc- and        His-tags    -   Regeneration buffer: 10 mM glycine pH 1.5    -   NRP2 ligands: Human SEMA3F-Fc (R&D Systems #9878-S3) VEGF-C(R&D        Systems #9199-VC/CF)

TABLE E3 Antibody Characterization Binding NRP2 Affinity Ligand BlockingEpitope 293Expi- by SPR: Fc-HRS(2-60) Domain NRP2 purified 293Expi-VEGF-C SEMA 3F Clone (ELISA) cells Ab (nM) NRP2 cells SPR SPR SPR 18138a2 Yes 27 Blocked Blocked Not Blocked aNRP2-14 blocked 8E2 b1 Yes 0.4Blocked Blocked Blocked Blocked aNRP2-6 5H11 b1 Yes 0.3 Blocked BlockedBlocked Blocked aNRP2-7 7G10 b1 Yes 1.4 Blocked Blocked Blocked BlockedaNRP2-8 9E7 b1 Yes 8 Blocked Blocked Blocked Blocked aNRP2-9 1E3 b1 Yes0.8 Blocked Blocked Blocked Blocked aNRP2-10 19E8 b1 Yes 2.7 BlockedBlocked Blocked Not aNRP2-15 Blocked 17F7 b2 Yes 41.8 Blocked PartialNot Not aNRP2-1 blocked blocked 13D7 b2 Yes 0.1 Blocked Blocked PartialNot aNRP2-11 blocked 3F2 c Yes 1.7 Not Not Not Not aNRP2-2 blockedblocked blocked blocked 20F3 c Yes 17.3 Not Not Not Not aNRP2-12 blockedblocked blocked blocked

The results demonstrate that even among different a2, b1, b2 and cdomain antibodies there are significant differences with respect toligand blocking as determined by SPR. Importantly, the antibodies showsignificant specific with respect to the selectivity of blocking VEGF-C,Semaphorin 3F, and HRS polypeptide binding. For example, and withoutlimitation, antibody clone18B8 (aNRP2-14) shows the ability toselectively block Sema 3F and HRS binding without blocking VEGF-Cbinding, and antibody 19E8 (aNRP2-15) shows the ability to selectivelyblock VEGF-C without blocking Sema 3F binding.

Example 3 Comparison of Antibody Binding Characteristics to CommerciallyAvailable Antibodies Using Wild Type A549 Cells Natively ExpressingHuman NRP2 Compared to NRP2 Null Cells

Additionally, the specificity of the in-house anti-NRP2 antibodiesbinding to NRP2 was validated using A549 wildtype versus A549 NRP2knockout clonal cells. The NRP2 knockout clonal cells were generated byCRISPR-Cas9 knock out, selection of single colonies, and validation forno NRP2 expression by western blots and cell surface immunostaining,following literature methods. Flow cytometry analysis was conductedessentially as described in Example 4 below. These in-house anti-NRP2bound to A549 wildtype cells while showed no or little binding to theNRP2 knockout clonal cells (see Table E4).

TABLE E4 Comparison of signal to noise binding characteristics comparedto commercially available antibodies Fold change of a-NRP2 toCtl{circumflex over ( )} (FC) Ab*/ Domain NRP2 NRP2 KO/ CloneSpecificity A549 WT KO_107 KO_235 WT % 18B8 a2 232.8 1.1 1.1 0.5aNRP2-14 19E8 b1 521.3 1.1 1.0 0.2 aNRP2-15 1E3 b1 410.2 1.6 1.4 0.4aNRP2-10 5H11 b1 397.6 1.5 1.4 0.4 aNRP2-7 7G10 b1 390.9 1.2 1.1 0.3aNRP2-8 8E2 b1 431.5 1.3 1.2 0.3 aNRP2-6 9E7 b1 461.9 1.2 1.1 0.2aNRP2-9 13D7 b2 286.6 1.2 1.5 0.5 aNRP2-11 17F7 b2 246.6 1.5 1.3 0.6aNRP2-1 20F3 c 61.1 1.2 1.4 2.1 aNRP2-12 3F2 c 348.9 2.1 2.0 0.6 aNRP2-2Commercial mabs R&D systems  23-857 57.6 5.4 5.4 9.4 #AF567   BAF2215 25-855 137.2 8.6 7.3 5.8 Boster C9-PE 560-858 6.0 1.7 1.7 27.9 SantaCruz hNRP2 Biotech C9-AF647 560-858 2.6 1.8 1.9 71.8 Santa Cruz hNRP2Biotech C9-AF647 560-858 1.8 1.3 1.7 — Santa Cruz hNRP2 Biotech CS #3366Mouse N- 0.9 1.0 1.4 — terminus

These results demonstrate that the claimed anti-NRP2 antibodies exhibitsignificantly superior specificity, and sensitivity to native NRP2expressed on a cell line, compared to existing commercially availableantibodies. These results are consistent with the idea that immunizationof animals with NRP2 expressing cell lines results in the generation ofan antibody response to the native NRP2 (non-denatured protein), whichmore accurately reflects the native conformation of the NRP2 proteinfound in vivo. As a result, the claimed antibodies would be anticipatedto exhibit superior biological activity compared to antibodies raisedrecombinant proteins, as described further below.

Example 4 Binding of Anti-Human Neuropilin 2 Antibodies to Human andCynomolgus Monkey NRP2 Overexpressing Cells

To assess the cross reactivity of the anti-NRP2 antibodies to human andcynomolgus monkey NRP2. NRP2 was expressed on the surface of HEK 293cells, and flow cytometry studies were conducted as more fully describedbelow. Establishing cross reactivity between cynomolgus monkey and humanNRP2 is an important therapeutic development consideration to ensurethat potential therapeutic candidates can be readily assessed fortoxicity in animal studies; particularly if there is little or no crossreactivity of the antibodies between human and rodent NRP2.

Purification of plasmid DNA.

Plasmids containing human or cynomolgus monkey NRP2 were purchased fromOrigene. To generate a large stock of plasmids and purify the DNA, eachplasmid was transformed into chemically competent E. coli cells (OneShot TOP10) according to the manufacturer's instructions. Thetransformed bacteria were grown in the recommended liquid medium (DifcoDehydrated Miler Luria-Bertani medium powder resuspended in water)containing kanamycin monosulfate (50 μg/mL) as an antibiotic. Theplasmid DNA was then purified using the QIAGEN DNA maxi prep kitaccording to the kit's instructions. DNA concentration and purity weremeasured on a spectrophotometer (Nanodrop 2000). An A260/A280 absorbanceratio between 1.8 and 2.0 was required for transfection.

Expi293 Propagation and Transient Transfection.

Expi293 cells, a HEK293 transient expression system maintained insuspension cultures, were used to express cynomolgus monkey NRP2. Cellswere grown in 60 mL Expi293 medium within 250 mL vented suspensionculture flasks. The propagation of cells was carried out in a MultitronCell incubator at 37° C., with 8% CO2, 80% humidity, and shaking at 225rpm. During the maintenance and expansion phase, Expi293 cells weresplit twice a week at 0.3e6 cells/mL in order to keep the density withinthe optimal range for transfection and the viability high. Celldensities and viabilities were determined using a cell counter (CedexHiRes Cell Analyzer).

The day prior to DNA transfection, the Expi293 cells were seeded at adensity of 2.0e6 cells/mL in order to maintain high viability (>95%) andlow density (not to exceed 3-5e6 cells/mL), thereby providing cells withfresh nutrients and avoiding transfection impediments caused by anysecreted substances.

Prior to DNA transfection, Expi293 cells were counted and reseeded at2.5e6 cells/mL in 50 mL shake flasks (TPP TubeSpin bioreactor tubes).The transfection procedure was performed using the Expifectamine kit.The manufacturer's protocol was adapted for a transfection in a totalvolume of 5 mL instead of 30 mL (i.e., all volumes were divided by afactor of 6). Five μg of each plasmid DNA was diluted in Opti-MEMreduced serum medium, complexed with Expifectamine transfection reagent,and transfected into Expi293 cells. The transfected pools were culturedwith shaking at 225 rpm to ensure complete suspension of the cellswithin the 50 mL bioreactors. Following the manufacturer's protocol,Expifectamine kit enhancers were added after 16-18 hours, and thecultures analyzed by flow cytometry 2 days post-transfection.

Generation of Expi293-hNRP2 Clonal Cells Stably Over-Expressing HumanNRP2.

A plasmid (Origene Technologies Cat#RC220706) encoding the human NRP2variant 2 transcript NM_003872 (hNRP2) fused to a Myc-DDK tag waspurchased. The vector was PCR amplified using Q5 polymerase (New EnglandBiolabs Cat#M0491) with the following primer pairs:

(SEQ ID NO: 125) 5′-TGAGGATGACAAAGATTTGCAGCT-3′ (SEQ ID NO: 126)5′-ACCGCGGCCGGCCGTTTATGCCTCGGAGCAGCACTT-3′ (SEQ ID NO: 127)5′-AGTGCCAAGCAAGCAACTCAAA-3′ (SEQ ID NO: 128)5′-AAGTGCTGCTCCGAGGCATAAACGGCCGGCCGCGGT-3′

The resulting PCR products were then fused, cut with MfeI/AgeI (NewEngland Biolabs Cat#R3589, R3552), and ligated into a vector fragment ofRC220706 cut with the same enzymes. This vector, containing an untaggedhNRP2 transcript, was then linearized and re-suspended in 10 mM Tris-0.1mM EDTA. Suspension Expi293 cells (ThermoFisher Cat#A14527), were grownin expression medium (ThermoFisher Cat#A1435101) at 37° C. and 8% CO2.The linearized plasmid described above was transfected into Expi293cells using an SF Cell Line 4D-Nucleofector® X Kit L (LonzaCat#V4XC-2012) and standard protocol T-030 for suspension HEK293 cells.Cells were allowed to recover in static culture for 17 hours,transferred to suspension and recovered an additional 72 hours, and thenwere selected with 200-350 μg/mL G418 in 50 μg increments (ThermoFisherCat#10131035). Cell densities and viabilities were monitored for aperiod of 3 weeks, with fresh media/antibiotic replacement every 2-3days.

To select hNRP2 over-expressing clonal cells, cells were suspended into96-well plates by limited dilution. Single colonies were transferred tofalcon tubes for further expansion under maintained selection pressure.Clonal cells that over-express human NRP2 were validated by a-NRP2staining followed with flow cytometry analysis.

Binding of a-NRP2 to Expi293-hNRP2 Clonal Cells or Cynomolgus MonkeyNRP2-Transfected Expi293 Cells.

Expi293-hNRP2 clonal cells or cynomolgus monkey (cyno) NRP2-transfectedExpi293 cells were collected by centrifugation at 300 g for 5 minutes,and washed twice by DPBS with calcium and magnesium (ThermoFisherCat#14040133). Washed cells were added to a 96-well V-bottom plate(ThermoFisher Cat#1424572) at 100,000 cells/well in 25 μL of DPBS withcalcium and magnesium. 25 μL Zombie Violet viability stain (BiolegendCat#423114, diluted at 1:250) was added to each well except the no-staincontrol. From this step on, cells were protected from light. Cells werekept at room temperature for 10 min and pelleted down at 300 g for 5minutes at 4° C. Supernatants containing Zombie Violet were discarded,and without washing, 30 uL of antibodies diluted in the flow wash buffer(FWB, DPBS with calcium and magnesium plus 2% FBS) were added to thecells. For a-NRP2 staining on Expi293-hNRP2 clonal cells, in-house mouseanti-NRP2 antibodies were tested at final concentrations of 0.05-30 nMor 0.02-10 nM at 3-fold dilutions. For a-NRP2 staining on CynoNRP2-transfected Expi293 cells, in-house a-NRP2 antibodies were testedat final concentrations of 0.01-200 nM at a 5-fold dilution. For eachtest, NRP2 over-expression on the cell surface was validated by a-NRP2staining (in-house aNRP2-2v2-1327 at 5 μg/mL and/or 10 μg/mL R&D #AF567a-NRP2) in separate wells. Binding was allowed to proceed on ice for 1hour. Cells were then pelleted at 300 g for 5 minutes at 4° C.,supernatants removed, and cells washed twice by adding 150 μL of FWB andcentrifuging the cells again in the same conditions. To detect a-NRP2binding on the cell surface, AF647-conjugated goat anti-mouse IgG orAF647-conjugated donkey anti-goat IgG (Jackson ImmunoResearchCat#115606062 & 705605147) was added to the cells at a finalconcentration of 2.5 μg/mL in 30 μL of FWB. After 30-45 minutes ofincubation on ice, cells were pelleted at 300 g for 5 minutes at 4° C.and supernatants removed, and the cells were washed twice as describedpreviously. Cells were then resuspended in FWB before acquisition on theCytoflex. Gains were set based on staining controls. Cells werecollected with typically more than 10,000 events. Cells were thenanalyzed on FlowJo analysis software by excluding dead cells.

As additional controls, the following conditions were tested: 1) Anisotype control mouse IgG1 (Biolgend Cat#400102) was used instead of themouse anti-NRP2 antibody to demonstrate the specificity of the staining;and 2) Staining with aNRP2-2v2-1327 or R&D #AF567 a-NRP2 was alsodetermined using mock- and non-transfected Expi293 cells to demonstrateover-expression of human or Cyno NRP2 on Expi293 cells. The statisticalanalysis was performed using GraphPad Prism. A four-parametervariable-slope curve was fitted to the data ([agonist] vs. response)using non-linear regression, and the EC₅₀ and r2 for each curve wasdetermined.

The EC₅₀ of four anti-NRP2 antibodies, each against a distinct domain ofNRP2, were measured for binding to both human and Cyno NRP2-transfectedExpi293 cells (Table E5). They all showed specific binding to NRP2 withEC₅₀₅ in the nanomolar range. By contrast, no binding was observed bythe isotype control mouse IgG1 antibody.

TABLE E5 Comparison of binding affinity to human and cynomolgus monkeyNRP2 Antibody Domain EC50 EC50 clone Specificity Human (nM) Cyno (nM)18B8 a2 0.9 5.5 aNRP2-14 1E3 b1 0.4 1.2 aNRP2-10 13D7 b2 0.9 4.5aNRP2-11 3F2 c 2.0 1.8 aNRP2-2 Isotype control n.d n.d No bindingantibody

The results from these studies demonstrate that each of the testedantibodies showed comparable binding affinities to human and cyno NRP2,which affinities that were all low nanomolar and within about 5-6 foldof each other between species.

Example 5 Binding of Anti-Human Neuropilin 2 Antibodies to CellsNatively Expressing NRP2

To evaluate binding of the anti-NRP2 antibodies to natively expressingcells, flow cytometry was conducted on a variety of cell lines known toendogenously express NRP2. To initially assess NRP2 expression levelswestern blots were conducted using 6 μg total cell lysate, and probedwith the commercially available anti-NRP2 antibody BAF2215 (BosterBiological Technology, California, USA), following standard methodology.Cell lines tested included U251 (Glioblastoma), A549 (lung cancer),HUVECs (human umbilical vein cells), THP-1 (human macrophage cell linepre-differentiated towards an M1 phenotype), and HLEC (Human lymphaticendothelial cells). Flow cytometry analysis was conducted essentially asdescribed in Example 4. Western blot results are shown in FIG. 3, andflow cytometry results are shown in FIGS. 4 and 5. Flow cytometrybinding data is also summarized in Table E6 (below).

TABLE E6 BINDING OF ANTI- HUMAN NEUROPILIN 2 ANTIBODIES TO CELLSNATIVELY EXPRESSING NRP2. Tested NRP2 level a-NRP2 cell surface bindingEC50 (nM) Cell Ab range 3F2 vs Ctl 18138 1E3 13D7 3F2 Isotype Type (nM)FC* aNRP2-14 aNRP2-10 aNRP2-11 aNRP2-2 control U251 0.01-1000 214.6 8.91.7 7.4 2.2 No binding HUVEC 0.06-1000 21.9 Not 27.1 0.4 30.3 Nosaturated binding HLEC 0.06-1000 8.6 n.d n.d n.d n.d n.d THP-1M10.01-1000 33.7 8.4 0.1 4.2 3.3 No binding THP-1M0 0.01-1000 3.0 n.d n.dn.d n.d n.d THP-1 0.01-1000 25.2 n.d n.d n.d n.d n.d WT A549 0.01-100025.2 Not Not 6.3 2.3 No saturated saturated binding

The results of these studies show that the anti-NRP2 antibodies showgood specificity for a wide range of cells that endogenously expressNRP2. The results confirm that the tested antibodies show highsensitivity to human NRP2 endogenous expressed on the cell surface on abroad range of cell types including cells derived from neuronal,epithelial, immune, lung, and cancer lineages.

Example 6 Ligand Displacement Studies with Cells Overexpressing NRP2

To assess the ability of the anti-NRP2 antibodies to displace thebinding of VEGF-C, Semaphorin 3F and HRS polypeptides, flow cytometrywas used. Binding of recombinant human VEGF-C(R&D Cat#9199-VC-025,untagged), Sema3F-Fc (R&D Cat#9878-S3-025, Fc chimera protein) orSema3F-p95m (prepared In-house), Myc-tagged Sema3F-p95 R583A R586A-1359)to Expi293-hNRP2 cells over expressing human NRP2 were determined byimmunostaining assay as described in Example 4. Studies were conductedwith cells incubated with each of these proteins at 0.05-100 nM at3-fold dilutions on ice for 1 hour. Cells were then washed and stainedwith secondary and/or detection antibodies as described previously.VEGF-C bound on the cell surface were stained by a rabbit anti VEGF-Cantibody (Abcam Cat#AB9546), followed with the detection by anAF647-conjugated goat anti-rabbit IgG (Jackson ImmunoResearchCat#111605144). Sema3F-Fc was detected by a Cy3-conjugated goatanti-human Fc IgG (Jackson ImmunoResearch Cat#109165098). Sema3F-p95mwas detected by an AF555-conjugated anti-Myc antibody (ThermoFisherCat#MA1980A555).

Binding curves were fit and EC₅₀ values determined as described above.VEGF-C was determined to bind to Expi293-hNRP2 cells with an EC₅₀ of 0.6nM. Sema3F-Fc was determined to bind to Expi293-hNRP2 cells with an EC₅₀of ^(˜)8.2 nM, and Sema3F-p95m bound to Expi293-hNRP2 cells with an withEC₅₀ of 5.7 nM.

For anti-NRP2 blocking of VEGF-C binding, Expi293-hNRP2 cells were firstincubated with anti-NRP2 for 30 min on ice. Without washing, VEGF-C wasadded at 0.6 nM to the cells. After 1 hour of incubation on ice, cellswere washed and then incubated with the rabbit anti VEGF-C antibody,followed with the detection by the AF647-conjugated goat anti-mouse IgGas described above. For anti-NRP2 blocking of Sema3F binding,Expi293-hNRP2 cells were first incubated with anti-NRP2 for 30 min onice. Without washing, Sema3F-p95m was added at 6 nM to the cells. After1 hour of incubation on ice, cells were washed and then incubated withthe AF555-conjugated anti-Myc antibody as described above.

Statistical analysis was performed using GraphPad Prism. Afour-parameter variable-slope curve was fitted to the data ([Inhibitor]vs. response) using non-linear regression, and the IC₅₀ and r2 for eachcurve was determined.

The tested anti-NRP2 showed different capabilities in blocking of VEGF-Cor Sema3F-p95m binding to Expi293-hNRP2 cells, and were categorized asblockers (>90% inhibition), partial blockers (30-90% inhibition), ornon-blockers (no obvious inhibition). Table E7 shows a summary of thebinding data, including the calculated IC₅₀ values (within subnanomolarto nanomolar range) for these anti-NRP2 antibodies, while FIG. 6 showsthe binding curves of the anti-NRP2 antibodies to Expi293-hNRP2 cellsover expressing human NRP2 in the absence of added ligand. FIG. 7 showsthe binding curves and FACS plots of the binding of human VEGF-C toExpi293-hNRP2 cells over expressing human NRP2. FIG. 8 shows theblocking and or displacement of VEGF-C binding to Expi293-hNRP2 cellsover expressing human NRP2. FIG. 9 shows the binding of Sema3F-p95 andSema 3F-p65 (0.05-100 nM) binding to Expi293-hNRP2 clonal cells overexpressing human NRP2. FIGS. 10 and 11 shows anti-NRP2 antibodyblocking, displacement curves for the indicated antibodies on Sema3F-p95 binding to Expi293-hNRP2 clonal cells over expressing human NRP2.

TABLE E7 LIGAND DISPLACEMENT STUDIES WITH CELLS OVEREXPRESSING NRP2Epitope Binding Block ligand binding to 293F-hNRP2 Domain 293F-hNRP2VEGF-C Fc-HRS(2-60) SEMA3F Clone (ELISA) (EC₅₀ nM) (IC₅₀ nM) (IC₅₀ nM)(IC₅₀ nM) 1868 a2 Binding 0.9 Partial 6.7 Not N/A Blocked 2.2 aNRP2-14blocked 8E2 b1 N.D. N.D. Blocked 1.1 Blocked N.D. N.D. N.D. aNRP2-6 5H11b1 N.D. N.D. Blocked 0.8 Blocked N.D. N.D. N.D. aNRP2-7 7G10 b1 Binding0.3 Blocked 0.3 Blocked 0.3 Partial 2.7 aNRP2-8 9E7 b1 Binding 0.7Blocked 0.7 Blocked 0.2 Partial 0.3 aNRP2-9 1E3 b1 Binding 0.3 Blocked0.4 Blocked 0.5 Partial 0.4 aNRP2-10 19E8 b1 Binding 0.3 N.D. N.D. N.D.N.D. Partial 0.4 aNRP2-15 17F7 b2 N.D. N.D. Not N /A N.D. N.D. N.D. N.D.aNRP2-1 blocked 13D7 b2 Binding 0.9 Blocked 5.8 Partial 34.3 Partial 0.2aNRP2-11 3F2 c Binding N.D. Not N/A Not N/A Not N/A aNRP2-2 blockedblocked blocked 20F3 c N.D. N.D. Not N/A N.D. N.D. N.D. N.D. aNRP2-12blocked

The results from these studies show specific and selective binding ofspecific antibodies with respect to displacing the binding of VEGF-C,Semaphorin 3F and HRS polypeptides. Importantly these results validateand extend the initial ligand displacement capacity of these bodies inthe context of NRP2-ligand interactions in the native cellularenvironment. For example, the results show that clone 18138 (aNRP2-14)shows the ability selectively block Semaphorin binding, while onlypartially blocking VEGF-C binding, and HRS polypeptides binding, andthat clones 7G10 (aNRP2-8), 9E7 (aNRP2-9), and 1E3 (aNRP2-10) shows theability selectively block VEGF-C and HRS polypeptides while onlypartially blocking binding Semaphorin binding. Clone 13D7 (aNRP2-11)shows the ability selectively block VEGF-C, while only partiallyblocking binding Semaphorin binding and HRS polypeptides binding.Whereas clone 3F2 (aNRP2-2) showed the ability to bind to NRP2 withoutdisplacing any of the tested ligands—making it particularly useful as adiagnostic reagent for NRP2 expression, particularly for example in IHCapplications.

Example 7 Characterization of Anti-NRP2 Antibodies on Receptor NRP2Dimerization

To further extend the assessment of the biological activity of theanti-NRP2 antibodies, their activity was assessed in a receptordimerization assay. In brief, vectors encoding a split luciferasepBiT1.1 and pBiT2.1 were obtained from Promega corporation. The completeextracellular domain and transmembrane helices of NRP2v2, FLT4 (VEGFR3),and plexin A1 (PLXNA1) were cloned into the vectors and screened foroptimal orientation, following established methods. Expi293 cells(Fisher) were transfected at 1 million cells/mL ^(˜)20 hours prior tothe assay with NRP2v2 and a co-receptor at equal mass amounts. Cellswere counted, and 100,000 live cells were plated in a well of a whiteluminometer plate in Optimem media (Fisher). Nano-Glo assay substrate(Promega) was added, and the plate was read on a MicroBeta luminometerat room temperature to establish baseline luminescence. Antibodies wereadded to the well at 100 nM and the plate was again read to establish anew baseline and monitor the effects of the antibodies on spontaneousreceptor dimerization. Either SEMA3F-p95 (aTyr pharma) or VEGF-C(R&Dsystems) were then added, at 200 nM or 20 nM respectively and the platewas again read to measure dimerization of receptors. Raw response isshown for NRP2/PLXNA1 dimerization, due to weak response and highvariability NRP2/FLT4 was normalized to the baseline prior to theaddition of ligand. Results are summarized in Table E8 below, andgraphically in FIGS. 12 and 13.

TABLE E8 Receptor Dimerization blocking summary: Domain Ability to BlockAbility to Block Antibody specificity NRP2/PLXNA1 NRP2/FLT4(VEGFR3)aNRP2-14 a2 Very strong None aNRP2-9 b1 Partial Partial aNRP2-10 b1Weak/None Very strong aNRP2-1 b2 Partial None aNRP2-11 b2 Strong Verystrong aNRP2-2 c Partial None aNRP2-12 c Strong Partial

The results demonstrate that all of the tested antibodies showfunctional activity in these assays. Surprisingly specific antibodiesshow extremely specific and non obvious functional differentiation. Forexample, antibody aNRP2-14 is demonstrated to exhibit extremely potentinhibition of NRP2-plexin heterodimerization, while having nosignificant impact on NRP2-VEGFR3 heterodimerization. Additionally,antibody aNRP2-10 shows the ability to potently inhibit NRP2-VEGFR3heterodimerization, without significantly inhibiting NRP2-plexinheterodimerization. By contrast antibody clone aNRP2-11 shows theability to strongly inhibit the heterodimerization of NRP2 to bothVEGFR3 and Plexin receptors. Further, antibody aNRP2-12 shows thesurprising ability to strongly inhibit the heterodimerization of NRP2 toplexin receptors, while only partially impacting NRP2-VEGFR3dimerization.

Example 8 Characterization of Antibody Fab and N NRP2 Complex CrystalStructure

Human NRP2 (aa25-595) with an N-terminal His-tag was expressed inExpi293 cells and purified by Ni-NTA affinity chromatography. aNRP2-14Fab light chain and His-tagged heavy chain were co-expressed in ExpiCHOcells and purified by Ni-NTA affinity chromatography. Bound complex ofNRP2 and aNRP2-14 Fab was obtained by mixing and purification by sizeexclusion chromatography. The protein complex was concentrated to 18.1mg/ml in 1×PBS pH 7.4. The proteins were screened for crystallizationhits by the sitting drop method (0.5 μL protein plus 0.5 μl precipitant)and incubated at 16° C. After 2-4 days of incubation, the complexcrystal grew in the condition containing 30% v/v polyethylene glycol 300and 0.1 M sodium acetate trihydrate pH 4.5. Crystals were cryoprotectedin the same conditions with the addition of 15% v/v glycerol beforebeing flash-cooled and stored in liquid nitrogen.

All the diffraction data were collected on a PILATUS3 6M detector at theShanghai Synchrotron Radiation Facility beamline BL19U, at 100 K, andindexed and processed with the HKL3000 software (Minor et al, 2006). Thestructure of the complex was determined by molecular replacement withPhaser (McCoy et al, 2007) in the CCP4 suite (Winn et al, 2011) usingpreviously published models as the search model [PDB code: NRP2 (2QQK),Fab (2D03)]. The final models were generated through multiple steps ofbuilding in Coot (Emsley et al, 2004) and refinement in Refmac(Murshudov et al, 2004) in the CCP4 suite. All the structure images andalignments were generated in PyMOL (DeLano, 2015). Buried surface areaswere calculated by the EBI PISA server(www.ebi.ac.uk/msd-srv/prot_int/cgibin/piserver).

Crystals of the NRP2/aNRP12-14 Fab had a space group of C2 with cellconstants of a=89.5 Å, b=89.4 Å, c=130.9 Å and diffracted to a maximumresolution of 1.90 Å. The structure was refined to 1.90 Å resolutionwith a final R_(work) and R_(free) of 19.71% and 23.46% respectively.While the Fab and the a2, b1 and b2 domain of NRP2 were well resolved(FIG. 14), Table E10, the electron density corresponding to the a1domain of the NRP2 protein could not be detected.

The aNRP2-14 antibody recognizes human NRP2 but not mouse NRP2. Amongthe NRP2 residues in the crystal structure that make contacts with theFab (FIG. 15, Table E9), the only residue that is not conserved betweenhuman and mouse is residue 237, which is a glutamic acid in human and alysine in mouse. This E237 residue in human NRP2 forms a hydrogen bondwith the S57 residue in the CDR2 region of the antibody heavy chain(FIG. 16), and therefore plays a key role in the binding interaction.

TABLE E9 Summary of interacting residues between NRP2 and aNRP2-14 FabNRP2 domain NRP2 residue Fab domain Fab residue Bond a2 D258 VL T32H-bond L192 VH Y105 H-bond T232 VH Y106 H-bond T234 VH S103, Y106 H-bondP235 VH S103 H-bond E237 VH S57 H-bond R263 VH T58, D59, Y105 H-bond VHD59 Salt bridge b1 E284 VH N74 H-bond S285 VH H-bond R287 VH G55,557H-bond N290 VH S32 H-bond D314 VH S77 H-bond

TABLE E10 Crystallographic data collection and refinement statisticsData collection Space group C2 Wavelength (Å)  0.97915 Unit cellparameters 89.501Å 89.411Å 130.877Å 90° 94.18° 90° Resolution range (Å)50-1.90 (1.93-1.90) No. of unique reflections   78778 (3849) Redundancy   5.9 (6.1) I/σ   23.3 (2.4) Completeness (%)   97.4 (96.1) R_(merge)^(a) (%)    7.4 (59.0) CC_(1/2) (highest-resolution shell)^(b)  0.816Structure refinement Resolution (Å)  1.90 R_(work) ^(c) (%) 19.71R_(free) ^(d) (%) 23.46 RMSD bonds (Å)  0.009 RMSD angles (°)  1.555Average B factor (Å²) 40.2 Ramachadran plot (%) Preferred 95.68 Allowed 3.58 Outliers  6 Numbers in parentheses represent the values for thehighest-resolution shell. ^(a)R_(merge) = Σ|I_(i)-</>Σ|I_(i), whereI_(i) is the intensity of measured reflection and <I> is the meanintensity of all symmetry-related reflections. ^(b)CC_(1/2) was definedin [4]. ^(c)R_(work) = Σ_(W)∥F_(calc)|-|F_(obs)∥/Σ|F_(obs)|, whereF_(obs) and F_(calc) are observed and calculated structure factors. W isworking dataset of about 95% of the total unique reflections randomlychosen and used for refinement. ^(d)R_(free) =Σ_(T)∥F_(calc)|-|F_(obs)∥/Σ|F_(obs)|, where T is a test dataset of about5% of the total unique reflections randomly chosen and set aside priorto refinement.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

1. A therapeutic composition, comprising at least one antibody orantigen-binding fragment thereof that specifically binds to a humanneuropilin-2 (NRP2) polypeptide (anti-NRP2 antibody), wherein the atleast one antibody or antigen-binding fragment thereof that specificallybinds to at least one epitope in a neuropilin domain selected from oneor more of the neuropilin A1 domain, neuropilin A2 domain, neuropilin B1domain, neuropilin B2 domain, neuropilin C domain, neuropilin A1/A2combined domain, neuropilin B1/B2 combined domain, neuropilin A2/B1combined domain, neuropilin B2/C combined domain, neuropilin A2/B1/B2combined domain, neuropilin A2/B1/B2/C combined domain, neuropilinA1/A2/B1 combined domain, neuropilin A1/A2/B1/B2 combined domain,neuropilin A1/A2/B1/B2/C combined domain, and the neuropilin B1/B2/Ccombined domain.
 2. The therapeutic composition of claim 1, wherein theat least one antibody or antigen-binding fragment thereof specificallybinds to the human NRP2 polypeptide in its native form but does notsubstantially bind to the human NRP2 polypeptide in its denatured form.3. (canceled)
 4. The therapeutic composition of claim 1, wherein the atleast one antibody or antigen-binding fragment thereof specificallybinds to at least one epitope in the neuropilin A1 domain, theneuropilin A2 domain, and/or the neuropilin A1A2 combined domain,including adjacent linker regions, optionally at about residues;(neuropilin A1 domain) 20-148, 30-141, 40-141, 50-141, 60-141, 70-141,80-141, 90-141, 100-141, 110-141, 120-141, 130-141; 20-130, 20-120,20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 asdefined by SEQ ID NO: 1 (FL human NRP2); (neuropilin A2 domain) 142-280,150-265, 160-265, 170-265, 180-265, 190-265, 200-265, 210-265, 220-265,230-265, 240-265, 250-265, 260-265, 141-270, 141-260, 141-250, 141-240,141-230, 141-220, 141-210, 141-200, 141-190, 141-180, 141-170, 141-160,141-150, 200-250, 210-250, 220-250, 230-250, 200-240, 210-240, 220-240,230-240, 227-247, 228-247, 229-247, 230-247, 231-247, 232-247, 233-247,234-247, 235-247, 236-247; 227-246, 227-245, 227-244, 227-243, 227-242,227-241, 227-240, 227-239, 227-238; 235-240, 236-239, 236-238, orresidue 237 as defined by SEQ ID NO:1 (FL human NRP2); or (combined A1A2domain) 20-280, 30-280, 40-280, 50-280, 60-280, 70-280, 80-280, 90-280,100-280, 110-280, 120-280, 130-280, 140-280, 150-280, 160-280, 170-280,180-280, 190-280, 200-280, 210-280, 220-280, 230-280, 240-280, 260-280,270-280, 20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-200,20-190, 20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110,20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 as defined bySEQ ID NO:1 (FL human NRP2). 5-17. (canceled)
 18. The therapeuticcomposition of claim 1, wherein the at least one antibody orantigen-binding fragment thereof modulates binding of the human NRP2polypeptide to at least one NRP2 ligand selected from: a VEGF selectedfrom one or more of VEGF-A145, VEGF-A165, VEGF-C, VEGF-D and PIGF-2; aVEGF receptor (VEGFR) selected from VEGFR2 and VEGFR3: a semaphorinselected from one or more of SEMA-3B, SEMA-3C, SEMA-3D SEMA-3F, andSEMA-3G; a plexin selected from one or more of plexin A1, A2, A3, A4,and D1; a growth factor selected from one or more of fibroblast growthfactor (FGF), hepatocyte growth factor (HGF), and platelet derivedgrowth factor (PDGF); a growth factor receptor selected from one or moreof a fibroblast growth factor receptor (FGFR), a hepatocyte growthfactor receptor (HGFR), and a platelet derived growth factor receptor(PDGF); a galectin or a galectin receptor a transcription factorselected from FAC1 and bromoprotein PHD finger transcription factor; anadaptor protein selected from one or more of GIPC1, GIPC2 and GIPC3; anintegrin selected from Table N3, optionally one or more of αvβ₁, αvβ₃,αvβ₅, αvβ₆, αvβ₈, α₆β₁ and α₆β₄; a transforming growth factor betaselected from one or more of TGFβ1, TGFβ2, TGFβ3, and theircorresponding TGFβ receptors; and an HRS polypeptide selected from TableH1, optionally an HRS splice variant selected from one or more ofHisRS^(N1), HisR^(N2), HisRS^(N3), HisRS^(N4)(SV9), HisRS^(N5),HisRS^(C1), HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5), HisRS^(C6),HisRS^(C7), HisRS^(C8)(SV11), and HisRS^(C9)(SV14). 19-26. (canceled)27. The therapeutic composition of claim 1, wherein the at least oneantibody or antigen-binding fragment thereof specifically binds to anepitope within the human NRP2 A2 domain which comprises at least 5contiguous amino acids of SEQ ID NO: 11, wherein the at least oneantibody or antigen-binding fragment thereof selectively inhibitsreceptor dimerization between NRP2 and plexin A1 without substantiallyinhibiting dimerization between NRP2 and FLT4 (VEGFR3).
 28. Thetherapeutic composition of claim 27, wherein the at least one antibodyor antigen-binding fragment thereof specifically binds to an epitopewithin amino acids 232-242 of human NRP2 SEQ ID NO:
 1. 29-34. (canceled)35. The therapeutic composition of claim 1, wherein the at least oneantibody or antigen-binding fragment thereof comprises a heavy chainvariable region (V_(H)) sequence that comprises complementarydetermining region V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences, and alight chain variable region (V_(L)) sequence that comprisescomplementary determining region V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences, wherein: the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequencescomprise SEQ ID NOs: 23-25, respectively, and the V_(L)CDR1, V_(L)CDR2,and V_(L)CDR3 sequences comprise SEQ ID NOs: 26-28, respectively,including variants thereof with 1, 2, 3, 4, or 5 alterations in theCDR(s) and which specifically bind to the human NRP2 polypeptide; theV_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs:29-31, respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3sequences comprise SEQ ID NOs: 32-34, respectively, including variantsthereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) and whichspecifically bind to the human NRP2 polypeptide; the V_(H)CDR1,V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs: 35-37,respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequencescomprise SEQ ID NOs: 38-40, respectively, including variants thereofwith 1, 2, 3, 4, or 5 alterations in the CDR(s) and which specificallybind to the human NRP2 polypeptide; the V_(H)CDR1, V_(H)CDR2, andV_(H)CDR3 sequences comprise SEQ ID NOs: 41-43, respectively, and theV_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequences comprise SEQ ID NOs:44-46, respectively, including variants thereof with 1, 2, 3, 4, or 5alterations in the CDR(s) and which specifically bind to the human NRP2polypeptide; the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences compriseSEQ ID NOs: 47-49, respectively, and the V_(L)CDR1, V_(L)CDR2, andV_(L)CDR3 sequences comprise SEQ ID NOs: 50-52, respectively, includingvariants thereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) andwhich specifically bind to the human NRP2 polypeptide; the V_(H)CDR1,V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs: 53-55,respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequencescomprise SEQ ID NOs: 56-58, respectively, including variants thereofwith 1, 2, 3, 4, or 5 alterations in the CDR(s) and which specificallybind to the human NRP2 polypeptide; the V_(L)CDR1, V_(H)CDR2, andV_(H)CDR3 sequences comprise SEQ ID NOs: 59-61, respectively, and theV_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequences comprise SEQ ID NOs:62-64, respectively, including variants thereof with 1, 2, 3, 4, or 5alterations in the CDR(s) and which specifically bind to the human NRP2polypeptide; the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences compriseSEQ ID NOs: 65-67, respectively, and the V_(L)CDR1, V_(L)CDR2, andV_(L)CDR3 sequences comprise SEQ ID NOs: 68-70, respectively, includingvariants thereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) andwhich specifically bind to the human NRP2 polypeptide; the V_(H)CDR1,V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ ID NOs: 71-73,respectively, and the V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequencescomprise SEQ ID NOs: 74-76, respectively, including variants thereofwith 1, 2, 3, 4, or 5 alterations in the CDR(s) and which specificallybind to the human NRP2 polypeptide; the V_(L)CDR1, V_(H)CDR2, andV_(H)CDR3 sequences comprise SEQ ID NOs: 77-79, respectively, and theV_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequences comprise SEQ ID NOs:80-82, respectively, including variants thereof with 1, 2, 3, 4, or 5alterations in the CDR(s) and which specifically bind to the human NRP2polypeptide; the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences compriseSEQ ID NOs: 83-85, respectively, and the V_(L)CDR1, V_(L)CDR2, andV_(L)CDR3 sequences comprise SEQ ID NOs: 86-88, respectively, includingvariants thereof with 1, 2, 3, 4, or 5 alterations in the CDR(s) andwhich specifically bind to the human NRP2 polypeptide.
 36. Thetherapeutic composition of claim 1, wherein the at least one antibody orantigen-binding fragment thereof comprises an IgA (including subclassesIgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3,and IgG4), or IgM Fc domain, optionally a human Fc domain, or a hybridand/or variant thereof.
 37. The therapeutic composition of claim 36,wherein the at least one antibody or antigen-binding fragment thereofcomprises an IgG Fc domain with high effector function in humans,optionally an IgG or IgG3 Fc domain.
 38. The therapeutic composition ofclaim 36, wherein the at least one antibody or antigen-binding fragmentthereof comprises an IgG Fc domain with low effector function in humans,optionally an IgG2 or IgG4 Fc domain.
 39. The therapeutic composition ofclaim 38, wherein the at least one antibody or antigen-binding fragmentthereof comprises an IgG1 or IgG4 Fc domain, optionally selected fromTable F1.
 40. The therapeutic composition of claim 1, wherein the atleast one antibody or antigen-binding fragment thereof is a monoclonalantibody, an Fv fragment, a single chain Fv (scFv) polypeptide, anadnectin, an anticalin, an aptamer, an avimer, a camelid antibody, adesigned ankyrin repeat protein (DARPin), a minibody, a nanobody, or aunibody.
 41. The therapeutic composition of claim 1, wherein the atleast one antibody or antigen-binding fragment thereof is a humanizedantibody.
 42. (canceled)
 43. The therapeutic composition of claim 1,wherein the composition has a purity of at least about 80%, 85%, 90%,95%, 98%, or 99% on a protein basis with respect to the at least oneantibody or antigen-binding fragment, and is substantiallyaggregate-free.
 44. The therapeutic composition of claim 1, wherein thetherapeutic composition is substantially endotoxin-free. 45-64.(canceled)
 65. A method of treating a disease or condition in a subjectin need thereof, comprising administering to the subject a therapeuticcomposition of claim
 1. 66. The method of claim 65, wherein the diseaseor condition is an NRP2-associated disease or condition.
 67. The methodof claim 65, wherein the disease or condition is selected from one ormore of cancer and diseases and pathways associated with cancer,including cancer cell growth, initiation, migration, adhesion, invasion,and/or metastasis; diseases associated with inflammation, autoimmunity,and related inflammatory diseases, including diseases associated withinappropriate immune cell activation or migration such as Graft versushost disease (GVHD); diseases associated with lymphatic development,lymphangiogenesis, and lymphatic damage, including, for example, edema,lymphedema, secondary lymphedema, inappropriate fat absorption anddeposition, excess fat deposition, and vascular permeability; diseasesassociated with infections, including latent infections; diseasesassociated with allergic disorders/diseases, allergic responses,including, for example, chronic obstructive pulmonary disorder (COPD),neutrophilic asthma, antineutrophil cytoplasmic antibody(ANCA)-associated systemic vasculitis, systemic lupus erythematosus,rheumatoid arthritis, inflammasome-related diseases, and skin-relatedneutrophil-mediated diseases such as pyoderma gangrenosum; diseasesassociated with granulomatous inflammatory diseases, includingsarcoidosis and granulomas; diseases associated with fibrosis includingfibrotic diseases, fibrosis, endothelial to mesenchymal transition(EMT), and wound healing; diseases associated with inappropriate smoothmuscle contractility, and inappropriate vascular smooth muscle cellmigration and adhesion; diseases associated with inappropriateautophagy, phagocytosis, and efferocytosis; diseases associated withinappropriate migratory cell movement; diseases associated with neuronaldiseases, peripheral nervous system remodeling, and pain perception; anddiseases associated with bone development and bone remodeling.
 68. Themethod of claim 65, wherein the disease is a cancer, optionally whereinthe cancer expresses or overexpresses NRP2, optionally wherein thecancer displays NRP2-dependent growth, NRP2-dependent adhesion,NRP2-dependent migration, and/or NRP2-dependent invasion.
 69. The methodof claim 68, wherein the cancer expresses or overexpresses NRP2 but doesnot substantially express neuropilin-1 (NRP1). 70-71. (canceled)
 72. Themethod of claim 65, comprising administering to the subject at least oneadditional agent selected from one or more of a cancer immunotherapyagent, a chemotherapeutic agent, a hormonal therapeutic agent, and akinase inhibitor. 73-74. (canceled)
 75. The method of claim 72, whereinthe cancer immunotherapy agent is selected from one or more of an immunecheckpoint modulatory agent, a cancer vaccine, an oncolytic virus, acytokine, and a cell-based immunotherapies. 76-92. (canceled)
 93. Themethod of claim 68, wherein the cancer is a primary cancer.
 94. Themethod of claim 68, wherein the cancer is a metastatic cancer,optionally a metastatic cancer that expresses NRP2 and/or NRP2B. 95-123.(canceled)
 124. The method of claim 67, wherein the disease is a diseaseassociated with vascular permeability, inappropriate wound healing,inappropriate smooth muscle contractility, inappropriate vascular smoothmuscle cell migration and adhesion, diseases associated withinappropriate migratory cell movement; diseases associated with neuronaldiseases, peripheral nervous system remodeling, and pain perception; anddiseases associated with bone development and bone remodeling.
 125. Abioassay system, comprising a substantially pure anti-NRP2 antibody orantigen-binding fragment thereof, as defined according to claim 1, and ahost cell line that expresses a human NRP2 polypeptide on the cellsurface. 126-141. (canceled)